In vivo delivery of nucleic acids to the liver or liver tissue

ABSTRACT

The invention encompasses methods of delivering nucleic acids, including dsRNA, to mammalian target cells in vivo via intercellular transfer, wherein the dsRNA is delivered to or expressed in a first cell different from the target cell, wherein the first cell facilitates delivery of the dsRNA to the target cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims benefit of priority tonon-provisional application Ser. No. 13/874,650 filed May 1, 2013 andSer. No. 13/874,654 filed May 1, 2013, and further claims the prioritybenefit of application Ser. No. 12/514,237 filed Sep. 2, 2010, now U.S.Pat. No. 8,524,679, issued Sep. 3, 2013, PCT international applicationPCT/US2007/083805 filed Nov. 6, 2007, provisional application Ser. No.60/857,501 filed Nov. 8, 2006, provisional application Ser. No.60/907,014 filed Mar. 16, 2007, provisional application Ser. No.60/956,610 filed Aug. 17, 2007, and provisional application Ser. No.60/974,695 filed Sep. 24, 2007, and provisional application Ser. No.60/978,950 filed Oct. 10, 2007, all which are incorporated herein byreference in their entireties.

FIELD OF INVENTION

The invention relates to methods of RNA-mediated inhibition of targetpolynucleotides in mammalian cells. This includes endogenously expressedsiRNA, shRNA, miRNA, antisense nucleic acids, and ribozymes as well asexogenously delivered synthetic siRNA, shRNA, miRNA, antisense nucleicacids and ribozyme molecules. The methods also apply to providingfunctional RNA including mRNA for gene therapy indications. Moreparticularly, it relates to methods of inhibiting the function of atarget polynucleotide in a target mammalian cell in vivo comprisingintroducing into a first cell such as a skin cell, muscle tissue cell,including skeletal or striated muscle cells (myocyte or myoblast), orany other competent cell of the mammal double stranded RNA (dsRNA) orpolynucleotide expression construct encoding one or more dsRNA or RNAiagent(s) including siRNA, shRNA, miRNA or other dsRNA, such that theRNAi agent is delivered distally to the target cell.

BACKGROUND OF THE INVENTION

The ability of double stranded RNA to effectively silence geneexpression, a phenomenon now commonly known as RNA interference (RNAi),has been one of the biggest scientific findings of the past decade.Recently, scientists Andrew Fire and Craig Mello were awarded the 2006Nobel Prize for Medicine for their pioneering work in the RNAi field.However, many challenges still remain to move RNAi from the laboratoryto the clinic. The biggest challenge to RNAi-mediated inhibition oftarget gene expression in animals and particularly humans is theefficient delivery of the RNAi agent to a sufficient number of targetcells. A variety of delivery mechanisms are currently being explored inthe RNAi field.

For instance, a number of groups have demonstrated successful andefficient delivery of double stranded (ds) RNA to mouse liver by tailvein injection. McCaffrey et al. (Nature Biotechnol. (2003) 21(6):639-44) report inhibiting production of hepatitis B virus replicativeintermediates in mice following tail vein injection of plasmidsexpressing HBV specific short hairpin RNAs (shRNAs). Giladi et al. (Mol.Therapy (2003) 8(5): 769-76) also report inhibition of HBV replicationin mice following tail vein injection of HBV specific short interferingRNA (siRNA), and Song et al. (Nature Med. (2003) 9(3): 347-51) reportRNA interference of fulminant hepatitis in mice following tail veininjection of siRNA specific for the fas gene.

While tail vein injection is suitable for inhibiting gene expression inmice, it is not a clinically relevant technique that may be used forhumans. However, several groups have also shown successful delivery ofdsRNA therapeutics without tail vein injection by systemic deliveryusing synthetic dsRNAs with improved stability. See Soutschek et al.Nature (2004) 432(7014): 173-8; see also Morrissey et al. Hepatol.(2005) 41(6): 1349-56. Local administration to the liver has also beendemonstrated by injecting double stranded RNA directly into thecirculatory system surrounding the liver using renal veincatheterization. See Hamar et al. PNAS (2004) 101(41): 14883-8. Stillothers have reported successful delivery of dsRNA and particularly siRNAusing cationic complexes or liposomal formulations. See, e.g., Landen etal. Cancer Biol. Ther. (2006) 5(12); see also Khoury et al. ArthritisRheumatol. (2006) 54(6): 1867-77.

In addition to injection into the circulatory system and lipid-basedmeans of delivering dsRNA, several groups have reported the use ofretroviral and adenoviral vectors for introducing dsRNA into mammals.For instance, Van den Haute et al. (Human Gene Therapy (2003) 14:1799-1807) report lentiviral vector delivery of short hairpin (shRNAs)against the reporter enhanced GFP (EGFP) that were shown to knock downgene expression of EGFP in mouse brain up to six months aftertransduction. McCaffrey et al. (Abstract No. 039, Keystone Symposia onsiRNAs and miRNAs, Apr. 14-19, 2004) report intravenous infusion ofrecombinant adenoviruses expressing HBV-specific shRNAs in HBV infectedmice as a possible treatment approach against hepatitis virus infectionin animals.

Thus, although some success has been shown using localized delivery, orby using systemic delivery of stabilized or complexed dsRNA, there isstill a great need for in vivo RNAi delivery mechanisms that do notrequire specialized formulations or invasive delivery procedures.Furthermore, the present inventors have shown that DNA-based endogenousdelivery of dsRNA is especially advantageous in allowing one to avoidthe interferon/PKR response while providing a prolonged supply ofexpressed dsRNA. See US 2004/0152117, which is herein incorporated byreference in its entirety. Accordingly, there is a particular need fortargeted delivery mechanisms for DNA-based RNAi expression vectors thatdo not require the use of viruses.

RNA interference was first discovered in the nematode C. elegans byNobel prize laureates Andrew Fire and Craig Mello and their colleagues.See U.S. Pat. No. 6,506,559, which is herein incorporated by reference.In U.S. Pat. No. '559, Fire and Mello et al. report that dsRNA-mediatedinhibition showed a surprising ability to cross cellular boundaries.This observation has since been described as a phenomenon that isparticular to nematodes or invertebrates, and corresponding modes ofsuch RNAi trafficking in vertebrate organisms have been generallydismissed.

The present inventors have surprisingly discovered, however, thatintramuscular, intradermal and subcutaneous delivery of expressionconstructs encoding dsRNA results in targeted inhibition of geneexpression in vivo in the liver and potentially other organs and tissuesof mammalian organisms. Without wishing to be bound by any theory, theinventors hypothesize that delivery of dsRNA to the liver from themuscle or skin, for example, may be mediated by extracellular vesicles(exovesicles) containing expressed RNA molecules such as dsRNA,antisense, miRNA, or mRNA or injected/introduced RNA molecules suchsiRNA, shRNA, etc. that bud from the surface of transfected musclecells. The extrusion of such exovesicles has been demonstrated forseveral cell types including muscle.

There is evidence in the art that certain lectins are exported frommuscle cells and myoblasts through evaginations of the cell membranewhich pinch off to form extracellular vesicles called exovesicles. Suchlectins including beta galectins are known to be on the surface of theextruded exovesicles. See Cooper and Barondes, Evidence of export of amuscle lectin from cytosol to extracellular matrix and for a novelsecretory mechanism. J. Cell Sci. (1990) 110: 1681-91; see also Harrisonand Wilson, The 14 kDa beta-galactoside binding lectin in myoblast andmyotube cultures: localization by confocal microscopy, J. Cell Sci.(1992) 101(Pt. 3): 635-46. Extracellular vesicles have also beenobserved at the periphery of fibroblasts, which are present in highquantity in the dermal layer of the skin. See Mehul and Hughes, 1997,Plasma membrane targeting, vesicular budding, and release of galectin 3from the cytoplasm of mammalian cells during secretion, J. Cell Sci.110: 1169-78. There is also evidence that lectins and certainglycoproteins may be cleared from the circulation by specific receptorson the surface of liver cells. See, e.g., Park et al., Theasialoglycoprotein receptor clears glycoconjugates terminating withsialic acid alpha 2,6GaINAc. PNAS (2005) 102(47): 17125-9; see alsoNagaoka et al., Galectin receptors are known to be expressed on thesurface of hepatocytes. Furthermore, betagalectin receptors have beenshown to be expressed in a polarized manner on the sinusoidal side ofthe hepatocytes, “A quantitative analysis of lectin binding to adult rathepatocytes cell surfaces”, In Vitro Cellular and Developmental Biology(1988) 24: 401-412; “Participation of a galectin-dependent mechanism inthe hepatic clearance of tissue-type plasminogen activator and plasmakallikrein.” Thromb Res (2003) 108: 257-262.

Thus, the present inventors propose that cytosolic content includingRNAs, e.g., mRNA, expressed siRNA/shRNA/miRNA, as well asinjected/introduced siRNA/shRNA/miRNA, or possibly even transfected DNApresent in the cytosol can be packaged within these exovesicles and betransported to distal sites such as the liver. Other mechanisms oftransfer have not been ruled out. Whatever the mechanism, to the presentinventors' knowledge, no one has recognized or proposed thatintramuscular, intradermal or subcutaneous administration of dsRNA andparticularly expressed dsRNA may be used in vivo in mammalian organismsas a therapeutic nucleic acid delivery mechanism for liver diseases aswell as diseases affecting other distal organs and tissues.

SUMMARY OF INVENTION

The present invention encompasses methods of delivering nucleic acids,including double stranded RNA molecules and/or polynucleotide expressionconstructs encoding RNA molecules, e.g., mRNAs, antisense, ribozyme ordsRNA including RNAi agents such as siRNA, shRNA, or miRNA, to a targetcell in vitro or ex vivo by delivering a nucleic acid to or expressing anucleic acid in a cell that is competent for distal cell targeting. Thecell may then be utilized for production in cell culture ofRNA-containing exovesicles, or for autologous or heterologous transplantinto a recipient mammalian organism. The cell may be a stem cell.

In one embodiment, among others, the invention includes methods ofdelivering at least one double stranded RNA (dsRNA) to a target cell inan animal comprising transfecting a first cell in the animal other thansaid target cell with a nucleic acid encoding said dsRNA, wherein saidtransfection results in the dsRNA being delivered to the target cell.The nucleic acid encoding the dsRNA may be cotransfected with a nucleicacid expressing a transmembrane or surface ligand specific for saidtarget cell, either on a single vector or via separate nucleic acidconstructs.

The methods of the present invention may be used to deliver any nucleicacid that is capable of being delivered from the transfected cell to adistal target cell. In some embodiments, the nucleic acid is apolynucleotide expression construct, e.g., a DNA plasmid or viralvector, which encodes an RNA effector molecule, e.g., an RNAi moleculecomprising a dsRNA region homologous and complementary to a target genein said distal organ or tissue (for instance, for mediating RNAinterference or RNAi), or another biologically active RNA. Suitableexpressed dsRNA or RNAi molecules include shRNA, siRNA and miRNA and aretypically between about 34 to about 500 bases in length and includedouble stranded or partially double stranded regions of at least about15 basepairs, typically 19 to 29 basepairs; mRNAs sizes typically rangefrom about 700 nts to about 15,000 nts in length.

In one embodiment, among others, the present invention encompassesmethods of delivering nucleic acids to distal organs and tissues viaintramuscular administration and transfection of muscle cells with atleast one type of nucleic acid in vivo. For instance, the inventionincludes a method of delivering at least one double stranded RNA (dsRNA)to a target organ or tissue in an animal comprising transfectingskeletal muscle cells in said animal with a nucleic acid encoding saiddsRNA, wherein said transfection results in said dsRNA being deliveredto said target tissue or organ. When skeletal muscle cells aretransfected, the target organ or tissue is an organ or tissue or cellother than skeletal muscle. In some embodiments, a skeletal muscle cellis transfected with an expression construct encoding a dsRNA molecule,the dsRNA molecule is expressed in the skeletal muscle cell, and thedsRNA molecule is delivered to a target cell that is not a skeletalmuscle cell.

In another embodiment, among others, the present invention encompassesmethods of delivering nucleic acids to distal organs and tissues viaintradermal or subcutaneous administration and transfection of skincells including fibroblasts with at least one type of nucleic acid invivo. For instance, the invention includes a method of delivering atleast one double stranded RNA (dsRNA) to a target organ or tissue in ananimal comprising transfecting skin cells in said animal with a nucleicacid encoding said dsRNA, wherein said transfection results in saiddsRNA being delivered to said target tissue or organ. When skin cellsare transfected, the target organ or tissue is an organ or tissue orcell other than the skin. In some embodiments, a skin cell istransfected with an expression construct encoding a dsRNA molecule, thedsRNA molecule is expressed in the skin cell, and the dsRNA molecule isdelivered to a target cell that is not a skin cell.

While some embodiments encompass transfecting skeletal muscle cells,skin cells or other competent cells in an animal with a nucleic acidencoding a dsRNA, methods wherein dsRNA is directly transfected intomuscle cells, skin cells or other competent cells are also encompassed.These dsRNA molecules include exogenously prepared transcribed andsynthetic siRNA, shRNA and or miRNA molecules, including chemicallymodified RNAs. For instance, the invention includes a method ofdelivering at least one dsRNA to a target organ or tissue in an animalcomprising transfecting skeletal muscle cells, skin cells or othercompetent targeting cells in said animal with said dsRNA, wherein saidtransfection results in said dsRNA being delivered to said other targetorgan or tissue. Suitable dsRNA molecules include shRNAs, siRNAs, andmiRNAs and are typically between about 34 to about 500 nucleotides,preferably comprising at least 15 to 29 basepairs in double-strandedconformation, including in some applications as miRNAs certainmismatches. The methods of the invention may be performed so as not totrigger an interferon/PKR response, for instance by using shorter dsRNAmolecules between 19 to 29 base pairs, or by using other methods knownin the art. See US Publication 2004/0152117, which is hereinincorporated by reference. The RNAs may be chemically modified as knownin the art to increase stability and decrease non-specific effects andtoxicity.

Applicants have also demonstrated that dsRNA molecules, including longdsRNA molecules (e.g., at least about 60, about 75, about 100, about150, about 200, about 300, about 400, about 500, about 600 bp andgreater), may be expressed intracellularly in stress-response competentmammalian cells (e.g., non-embryonic, differentiated or adult cells)without any evidence of their inducing an interferon, stress, or “panic”response, see US 2004/0152117Al, which is herein incorporated byreference in its entirety. In addition, the methods of the invention maythemselves serve to minimize or avoid triggering a stress response inthe target cell to which the dsRNA is delivered irrespective of thelength or nature of the dsRNA; e.g, dsRNAs delivered into distal cellswithin “blebs” may avoid triggering a stress response, in contrast tothe same dsRNAs entering through the cell membrane without the cover ofthe surrounding “bleb”. Any molecule that may be conjugated to dsRNA,co-transfected or co-expressed therewith, and delivered therewith to thetarget cell may be included.

The present invention also encompasses methods of treating or preventingdiseases in distal organs or tissues in an animal via intramuscular,intradermal or subcutaneous administration or transfection of othercompetent targeting cells, with at least one nucleic acid in vivo. Forinstance, the invention includes a method of treating or preventingdisease in a target organ or tissue in an animal, comprisingtransfecting skeletal muscle cells, skin cells or other competentnon-target cells in said animal with a nucleic acid encoding a dsRNAcorresponding to a target gene in a cell of said target organ or tissue,wherein said transfection results in said dsRNA being delivered to saidtarget tissue or organ, and wherein delivery of said dsRNA to saidtarget organ or tissue inhibits or reduces expression of said targetgene in said target organ or tissue thereby treating or amelioratingsaid disease. “Intramuscular”, “intradermal”, or “subcutaneous” deliveryas defined herein includes any method which achieves transfection ofcells within the identified tissue, including without limitation needleinjection into the tissue itself or delivery into a blood vessel whichsupplies the tissue, intravascular delivery into a vessel havingenhanced permeability, needleless injection, biolistic or gene gunprojection into a cell or tissue, any of the various knowninjection/electroporation technologies, transdermal patch, etc.

Administration may be via any method or device which can achieve thedesired introduction of polynucleotide, e.g., needle, syringe, catheteror cannula injection or needleless injection, e.g., a Bioject needlelessinjection device, which can be adjusted to deliver a liquid medicationto various depths including intradermal, subcutaneous, and/orintramuscular. A transdermal patch might also be employed, as well as abiolistic injector or “gene gun” device capable of shooting a plasmidexpression vector into cells. Any of the variousinjection/electroporation devices and technologies (Inovio/Genetronics,Ichor, VGX etc.) may also be used, as may hydrodynamic intravascularmethods which utilize various means, e.g., increasedpermeability/increased pressure, to promote delivery to cells includingmuscle, e.g., the Mirus Pathway IVT™ Gene Delivery methods (Mirus,Madison, Wis.) which utilize a cuff or tourniquet to restrict blood flowand increase pressure within the vessel in order to facilitateintravascular delivery of nucleic acids such as plasmid expressionvectors to tissues perfused by the vessel, e.g., limb muscle. A syringe,pump or other suitable device may be used to effect rapid intravasculardelivery while blood flow is transiently occluded, thereby promotingtransfection of the adjoining muscle cells.

The methods of the present invention are particularly suitable fortreating or preventing diseases or conditions of or involving the liverincluding viral diseases of the liver, liver cancer and genetic diseasesof the liver, or conditions which may be modulated by targeting an RNAor gene in the liver via e.g. antisense or RNA interference, or genetherapy where a functional mRNA is replaced. Expressed therapeutic mRNAsand proteins may also be delivered to target cells via the methods ofthe present invention. The methods are also appropriate for targetinggenes in the liver responsible for certain metabolic diseases ordisorders such as high cholesterol levels for example, including but notlimited to apolipoprotein B and pcsk9. The methods may also be used todeliver nucleic acid-based therapeutics to other cells and organs ortissues, including cancer cells or HIV-infected cells, for instance byexpressing suitable receptors or other ligands on the surface of targetcells, and/or by co-expressing cell surface or exovesicular-targetedligands in transfected competent targeting cells. The methods may alsobe used prophylactically, for instance to deliver dsRNAs or othernucleic acid-based therapeutics to target cells to protect againstfuture infection or disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Graph demonstrating the percent decrease in HBsAg expressionmediated by the NUC050 vector, which expresses four HBV-targeted shorthairpin dsRNAs, (experiment 183-9) compared to control vector (183-10)in NODsc^(scld) mice. Mice were administered either NUC050 plasmid(183-9) or negative control plasmid NUC049 (183-10), formulated withbupivacaine and injected intramuscularly (IM) at day 0. Both groups ofmice received a hydrodynamic injection (HDI) of HBsAg expression plasmidNUC054 at day −5. Serum sAg levels were measured at various days afterdosing. Values represent mean s Ag as a percent of the pre-bleed sAgvalue for each group of animals.

FIG. 2. Graph showing the ratio of Renilla:firefly luciferase (RLU) inresponse to muscle electroporation of NUC050 versus NUC049 in C57B1/6mice. Mice were administered either NUC050 plasmid or negative controlplasmid NUC049 via intramuscular (IM) injection concomitantly withelectroporation (EP) at day 0. On day 6, both groups of mice received ahydrodynamic injection (HDI) of the dual-luciferase reporter plasmidNUC060. Values represent the mean Renilla RLU/Firefly RLU ratio for eachgroup of animals. The 34.9% difference is statistically significant bynonparametric Wilcoxon two-sample test (p<0.05).

FIG. 3. Box-and-whisker plot of individual mouse ND-300 ratios forNUC050 versus NUC049. The distribution of Renilla:Firefly ratio valuesamong individual animals in each respective group can be visualized bythe box-and whisker plot. The ‘box’ portion represents the interquartilerange (Q1-Q3). The vertical line within the box represents the medianaverage.

FIG. 4. Schematic description of TaqMan RNA assays, TaqMan-basedreal-time quantification of siRNAs includes two steps, stem-loop RT andreal-time PCR. Stem-loop RT primers bind to the 3′ portion of miRNAmolecules and are reverse transcribed with reverse transcriptase. Then,the RT product is quantified using conventional TaqMan PCR that includesmiRNA-specific forward primer, reverse primer and dye-labeled TaqManprobes. The purpose of tailed forward primer at 5′ is to increase itsmelting temperature (Tm) depending on the sequence composition of miRNAmolecules.

FIG. 5. Photos of published muscle blebs stained immunologically forβ-galectin (FIG. 5, panels B, C and D) compared with a time lapsed photoby the present inventors of a myoblast secreting blebs (panel A).

FIG. 6. Box-and-whisker plot showing the distribution of Renilla:Fireflyratio values among individual animals in each respective group can bevisualized by the box-and whisker plot. The ‘box’ portion represents theinterquartile range (Q1-Q3). The vertical line within the box representsthe median average.

FIG. 7. Graph showing HBsAg Mean Values following intramuscularelectroporation of NUC050 plasmid. The mean sAg levels of the three dosegroups (10 ug, 5 ug, and 2 ug) are represented in graph (A) day +2, (B)day +7, and (C) day +16. The reduction in sAg in the NUC050 group issignificant (p<0.05) in the day +2 IO ug group, the day+7 IO ug group,and the day+7 5 ug group.

FIG. 8. Graph showing the ND-360 ratio of Renilla:Firefly luciferase(RLU) in response to subcutaneous delivery/administration of NUC050versus NUC049 in C57B1/6 mice. Mice were administered either NUC050plasmid or negative control plasmid NUC049 via subcutaneous (SC)injection at day 0. Both groups of mice received a hydrodynamicinjection (HDI) of the dual-luciferase reporter plasmid NUC060 at day 6.Values represent the mean Renilla RLU/Firefly RLU ratio in the liver foreach group of animals on day 11. The 16.7% difference is statisticallysignificant by nonparametric Wilcoxon two-sample test (p<0.05).

FIG. 9. Distribution of the individual mouse ND-360 ratios ofRenilla/Firefly

Luciferase activity for NUC050 versus NUC049. The distribution ofRenilla:Firefly activity ratios among individual animals in theexperimental (NUC050) and control (NUC049) groups of ND-360 is shown bythe box-and whisker plot. The ‘box’ portion represents the interquartilerange (Q1-Q3). The vertical line within the box represents the medianand the horizontal lines extend to the lower and upper extremes of thedistribution.

FIG. 10. Distribution of individual mouse normalized HBsAg levels forNUC 049 versus NUC050 at day 6 (13 days after vector injection) and day11 (18 days after vector injection).

DETAILED DESCRIPTION OF INVENTION

The present invention encompasses methods of delivering RNA includingdouble stranded RNA (dsRNA) to a distal target cell or organ or tissueby expressing the RNA (e.g., a dsRNA) in, or introducing the RNA into, afirst cell that is competent for inter-organ or inter-tissue orintercellular delivery. By “distal” is meant that the RNA, e.g., a dsRNAis transported to a different organ, tissue or cell than the cell intowhich it is originally introduced or expressed. “Competent forinter-organ or inter-tissue or intercellular delivery” or “competent fortargeting a distal cell or organ or tissue” means that the cell intowhich the dsRNA is expressed or introduced is able to facilitatedelivery of the dsRNA to a distal organ, tissue or cell, e.g., throughvesicle extrusion. Such cells include muscle cells and skin cells, andany other cell that is competent for RNA delivery to distal tissues.

The present invention is based on the surprising discovery thatintramuscular, intradermal or subcutaneous injection of a nucleic acidencoding a dsRNA corresponding to a target gene results in inhibition oftarget gene expression in the liver. Accordingly, in one embodiment, theinvention encompasses methods of delivering nucleic acids to distalorgans or tissues in vivo and methods of treating or preventing diseasesand disorders in distal organs or tissues via intramuscular, intradermalor subcutaneous administration and transfection of muscle or skin cellswith at least one dsRNA, or at least one nucleic acid expressing a dsRNAcorresponding to a target gene in a cell of said distal organ or tissue.

As reported herein, the present inventors have demonstrated thatintramuscular, intradermal or subcutaneous delivery of a vector or DNAconstruct expressing target-specific dsRNA molecules, e.g., shRNA (RNAi)molecules, is surprisingly able to reduce the expression of Hepatitis Bsurface antigen (sAg), as well as other target genes, in the liver.While not wishing to be bound by any mechanism, it is hypothesized thatthe encoded double-stranded RNA molecules (e.g., shRNAs or duplexdsRNAs) are transcribed from expression constructs transfected intomuscle or skin cells and the RNAi molecules are then delivered to theliver. While no evidence has been found that any significant quantity ofintact dsRNA expression plasmid DNA itself gets into liver hepatocytes,it cannot yet be ruled out that DNA encoding the double-strandedmolecules of the invention is transferred to the liver, either alone orin combination with the expressed dsRNA.

Muscle cells are one type of cell known to extrude plasma membranevesicles, or exovesicles, also known as membrane “blebbing”. Forexample, galectin is one protein expressed at high levels in skeletaland smooth muscle cells, which lacks a signal sequence and has beenshown to be secreted by a mechanism distinct from classical exocytosis.Prior to secretion, galectin has been observed to become specificallyconcentrated under the myoblast plasma membrane and in plasma membraneevaginations which appear to pinch off to form galectin richextracellular vesicles. Cooper and Barondes, 1990; Cooper, 1997,Galectin-1: Secretion and Modulation of Cell Interactions with Laminin,Trends in Glycoscience and Glycotechnol. 9(45): 57-67; Harrison andWilson, 1992, The 14 kDa β-galactosidase binding lectin in myoblast andmyotube cultures: localization by confocal microscopy, J. Cell Sci. 101:635-46.

Although the process of membrane blebbing and the extent to whichmembrane blebbing contributes to protein export in other cell types arestill unclear, membrane blebbing has also been observed in cells otherthan muscle cells. Indeed, a variety of different galectin genes havebeen detected in different cell types, each lacking a signal sequenceand containing a highly conserved core sequence of about 130 aminoacids. See Cooper and Barondes, 1999. Galectin-1, the best-characterizedof the galectin family, is expressed in many tissues besides skeletaland smooth muscle, including liver, lung, heart, spleen, intestine,brain, lymphocytes, thymocytes and other vascular cells, the olfactorysystem, and the central and peripheral nervous systems. Inagaki et al.2000, Oxidized galectin-1 promotes axonal regeneration in peripheralnerves but does not possess lectin properties, European J. Biochem.267(10): 2955-64. Galectin-1 is also expressed and secreted by CHO cellsby a non-classical mechanism. See Seelenmeyer et al., 2005, Cell surfacecounter receptors are essential components of the unconventional exportmachinery of galectin-1, J. Cell Biol. 171: 373-81. Galectin-3, whichhas also been shown to be secreted in exovesicles that pinch off theplasma membrane, has been detected in activated macrophages,eosinophils, neutrophils, mast cells, the epithelium of thegastrointestinal and respiratory tracts, the kidneys and some sensoryneurons as well as many tumors. Krzeslak and Lipinska, 2004, Galectin-3as a multifunctional protein, Cell. MoI. Biol. Letts. 9: 305-28.

Membrane blebbing, sometimes referred to as ectocytosis, has beenobserved at the periphery of many cell types, including fibroblasts,neutrophils and chondrocytes. See Mehul and Hughes, 1997, Plasmamembrane targeting, vesicular budding, and release of galectin 3 fromthe cytoplasm of mammalian cells during secretion, J. Cell Sci. 110:1169-78. Further, it has been hypothesized that shedding of membraneexovesicles may also be a mechanism for FGF-2 secretion from a varietyof other cell types. See Walter Nickel 2005 Unconventional SecretoryRoutes: Direct Protein Export Across the Plasma Membrane of MammalianCells, Traffic 6: 607-14. Also, membrane blebbing has also been proposedas a potential secretory mechanism for certain apocrine-synthesizedproteins, including secretory transglutaminase. See Aumuller et al. 1999Apocrine secretion-Fact or artifact? Ann. Anat. 181(5): 437-46. Assumingmembrane blebbing is involved in the effects reported in the presentinvention, the methods of the present invention may be performed usingany exovesicle-producing cell that is currently known or that will beidentified in the future. Since extruded exovesicles are on averageabout 80 nm in diameter and coated with surface galectins, they have thepotential to interact with cell types that contain galectin receptors.Such cells include hepatocytes, whose galectin receptors are positionedto face the sinusoids and thus be exposed to molecules and particles inthe blood.

Exovesicles, which generate as a process of membrane blebbing orshedding, should be distinguished from exosomes or multivesicularbodies. See Walter Nickel 2005; see also Cooper, 1997. Exosomes areproduced from the endoplasmic reticulum and have very little if anycytosolic content, although they do have some MHC molecules on theirsurface and can be used as antigen presenting vehicles. In contrast,exovesicles bud from the surface of muscle cells and include cytosoliccontent as has been demonstrated by the inventors. Indeed, in theprocess of expressed interfering RNA (eiRNA), transcription occurs inthe nucleus but the RNA is then transported to the cytoplasmiccompartment, where the inventors hypothesize it is then incorporatedinto exovesicles blebbing off the membrane surface.

Notwithstanding the actual mechanism, the present invention encompassesmethods of delivering at least one nucleic acid to a target cell in ananimal comprising transfecting a first cell in the animal other thansaid target cell that is competent for distal cell targeting with anucleic acid encoding an RNA of interest, e.g., an “RNA effectormolecule” having a desired biological activity, e.g., an antisense RNA,triplex-forming RNA, ribozyme, an artificially selected high affinityRNA ligand (aptamer), a double-stranded RNA (e.g., siRNA, shRNA, miRNA)or other regulatory RNA or an mRNA, wherein said transfection results ineither the DNA or the encoded RNA being delivered to the target cell.The RNA may or may not be polyadenylated. The RNA may or may not becapable of being translated. The first competent cell may also betransfected in vitro or ex vivo, and the competent cell thereafterintroduced into the animal. The methods of the present invention may beused to deliver any nucleic acid that is present in the cytoplasm of thetransfected cell and is capable of being delivered to the distal targetcell. In addition to transfected and expressed dsRNAs, the presentinvention also includes methods of delivering expressed mRNA fromtherapeutic genes. RNAs that may be delivered to the distal target cellinclude, but are not limited to, antisense RNA, ribozyme RNA, dsRNAsincluding hairpin dsRNA, microRNA and duplex dsRNA molecules, as well asmRNAs.

Suitable “competent” cells for use in the methods of the inventioninclude muscle cells, skin cells and any other competent cell capable ofdelivering nucleic acids to a distal target cell. Other competent cellsmay be identified using one of the RNA transfer assays disclosed in WO00/63364, which is herein incorporated by reference in its entirety. Forinstance, Example 2 of WO 00/63364 describes two different in vitroassays that may be used to detect the transfer of RNA molecules betweenco-cultured donor and target cells.

By “double stranded RNA” or “dsRNA” is meant a ribonucleic acidcontaining at least a region of nucleotides that are in a doublestranded conformation. The dsRNA may be a conventional siRNA, shRNA ormiRNA (including primary transcript or pri-miRNA, pre-miRNA, orfunctional miRNA) or an RNA that contains more than one hairpinstructure. The double stranded RNA may be a single molecule with one ormore region(s) of self-complementarity such that nucleotides in onesegment of the molecule base pair with nucleotides in another segment ofthe molecule. In various embodiments, a double stranded RNA thatconsists of a single molecule consists entirely of ribonucleotides, acombination of ribonucleotides and modified bases, or includes a regionof ribonucleotides that is complementary to a region ofdeoxyribonucleotides. Alternatively, the double stranded RNA may includetwo different strands that have one or more region(s) of complementarityto each other. Desirably, the region of the double stranded RNA that ispresent in a double stranded conformation includes at least about 15 to20, 20 to 25, 25 to 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000nucleotides participating in one strand of the double strandedstructure, or includes all of the nucleotides being represented in thedouble stranded RNA. In some embodiments, the double stranded RNA isfully complementary, and does not contain any single stranded regions,such as single stranded ends. In other embodiments, as e.g., miRNA-typedsRNA molecules, the double-stranded regions may be interspersed withone or more single-stranded nucleotides or areas. In some embodimentsthe dsRNA is an shRNA. All such synthetically prepared and exogenouslydelivered RNAs, including shRNAs and duplex or siRNAs, may be chemicallystabilized and chemically modified, using one or more of the methods andchemical modifications known to those of skill in the art. Suchmodifications which may be used in combination include sulfur chemistrymodification such as phosphorothioate linkages that make the drug moreresistant to degradation, T-O-methoxyethyl modifications, etc. See e.g.Chiu and Rana, siRNA function in RNAi: A chemical modification analysis,RNA (2003), 9:1034-1048. Cold Spring Harbor Laboratory Press, theteaching of which is incorporated by reference.

In some embodiments, the dsRNA region of the RNA molecule corresponds toa target gene in said target organ or tissue (for instance, formediating RNA interference or RNAi). In such instances, the dsRNA regionis preferably not translated, and is substantially homologous andcomplementary to a region of the target gene. Where the dsRNA is usedfor RNA interference, one strand of the dsRNA structure or region, i.e.,the antisense strand, will have at least about 70, 80, 90, 95, 98, or100% complementarity to a target nucleic acid, and the other strand orregion, i.e., the sense strand or region will have at least about 70,80, 90, 95, 98, or 100% identity to a target nucleic acid. In suchembodiments, the dsRNA is considered to be both substantially homologousand complementary to the target gene, meaning that the dsRNA need not beentirely identical and complementary to the target gene so long as it isstill effective to mediate sequence specific RNA interference. SuchdsRNAs will usually have a sequence of at least 19 contiguousnucleotides 100% complementary and homologous to a target nucleic acid.Preferred for RNAi applications are short hairpin dsRNA (shRNA)molecules and microRNA (miRNA) molecules. By shRNA (short-hairpin RNA)is meant an RNA molecule of less than approximately 400 to 500nucleotides (nt), preferably less than 100 to 200 nt, in which at leastone stretch of at least 15 to 100 nucleotides (preferably 17 to 50 nt,more preferably 19 to 29 nt) is base paired with a complementarysequence located on the same RNA molecule, and where said sequence andcomplementary sequence are separated by an unpaired region of at leastabout 4 to 7 nucleotides (preferably about 9 to about 15 nucleotides)which forms a single-stranded loop above the stem structure created bythe two regions of base complementarity. The shRNA molecules comprise atleast one stem-loop structure comprising a double-stranded stem regionof about 17 to about 100 bp; about 17 to about 50 bp; about 40 to about100 bp; about 18 to about 40 bp; or from about 19 to about 29 bp;homologous and complementary to a target sequence to be inhibited; andan unpaired loop region of at least about 4 to 7 nucleotides, preferablyabout 9 to about 15 nucleotides, which forms a single-stranded loopabove the stem structure created by the two regions of basecomplementarity. In addition to single shRNAs, included shRNAs can bedual or bi-finger and multi-finger hairpin dsRNAs, in which the RNAmolecule comprises two or more of such stem-loop structures separated bya single-stranded spacer region. A recombinant vector may be engineeredto encode multiple, e.g., three, four, five or more short hairpin dsRNAsand/or other RNAs such as mRNAs. The hairpin dsRNA may be a singlehairpin dsRNA or a bi-fingered, or multi-fingered dsRNA hairpin asdescribed in PCT/US03/033466 or WO 04/035766 or a partial or forcedhairpin structure as described in WO 2004/011624, or the dsRNA may be apolynucleotide comprising one or more dsRNA effector molecules encodedin a miRNA context as described in PCT/US2007/81103 filed 1 I-Oct.-2007.The teachings of each of these documents are incorporated herein byreference in their entireties.

An siRNA can be expressed or synthetic and is comprised of two RNAstrands that basepair with each other to form a dsRNA, i.e., duplex RNA.The basepairing does not need to be 100% and thus the complementarity ofone strand with the second does not need to be 100%. Complementarityshould be sufficient to maintain the stands in double-strandedconfirmation. The amount of complementarity needed is sequence andlength dependent and can be easily calculated by one skilled in the art.siRNA length is most optimally 19-29 bp, next preferred is 30-40 bp. Alimited number of mismatches within the double-stranded region,especially in the sense strand, is compatible with RNAi activity. siRNAsmay be chemically stabilized and chemically modified, using one or moreof the methods and chemical modifications known to those of skill in theart.

By “target nucleic acid” is meant the nucleic acid sequence in thedistal target organ, tissue or cell whose expression is modulated as aresult of sequence-specific nucleic acid based inhibition, e.g.,post-transcriptional or transcriptional gene silencing, antisenseinhibition, ribozymal cleavage, etc. The target nucleic acid sequencecan be any nucleic acid in the target cell, DNA, RNA or DNA/RNA hybrid,whose expression is desired to be modulated, including withoutlimitation gene sequences or chromosomal sequences endogenous to thecell as well as introduced sequences, genomic sequences, cDNA, mRNAsequences, sequences of intracellular pathogens such as viral nucleicacids present in the cell, transcribed and non-transcribed sequences,coding and non-coding sequences, translated and non-translated sequencesincluding 3′ and/or 5′ UTRs, and regulatory sequences such astranscription factor binding site, promoter, enhancer, and repressorsequences. Suitable target nucleic acid sequences are associated withcancer or abnormal cell growth, such as oncogenes, and nucleic acidsequences associated with an autosomal dominant or recessive disorders,as well as nucleic acid sequences associated with pathogens includingviruses. To “modulate” means to decrease the expression of a targetnucleic acid in a cell, or the biological activity of the encoded targetpolypeptide in a cell, by at least about 20%, more desirably by at leastabout 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or even 100%. In someinstances, expression of genes in the target cell may also be increased,for instance where the gene targeted by the dsRNA is a transcriptionalrepressor or other negative regulatory gene. In some instances thetarget nucleic acid will not be present in the first transfected cell.In some instances the target nucleic acid will be present in the firsttransfected cell as well as in the distal target cell.

Typically with expressed interfering RNA (eiRNA), the dsRNA is expressedin the first transfected cell from an expression vector. In such avector, the sense strand and the antisense strand of the dsRNA may betranscribed from the same nucleic acid sequence using e.g., twoconvergent promoters at either end of the nucleic acid sequence orseparate promoters transcribing either a sense or antisense sequence.Alternatively, two plasmids can be cotransfected, with one of theplasmids designed to transcribe one strand of the dsRNA while the otheris designed to transcribe the other strand. Alternatively, the nucleicacid sequence encoding the dsRNA comprises an inverted repeat, such thatupon transcription from a single promoter, the expressed RNA forms adouble stranded RNA, i.e. that has a hairpin or “stem-loop” structure,e.g., an shRNA. The loop between the inverted repeat regions, or senseand antisense regions, is typically at least four base pairs, but can beat least about 10, at least about 15, at least about 20, at least about25, at least about 30, at least about 50, or at least about 75, or more,or any size that permits formation of the double stranded structure.Multiple stem-loop structures may be formed from a single RNA transcriptto generate a multi-target dsRNA. See WO 00/63364, and WO2004/035765,which are herein incorporated by reference in their entireties. Hairpinstructures may be partial or forced hairpin structures as described inWO2004/011624, incorporated herein by reference.

By “expression vector” is meant a recombinant vector including a DNA orRNA construct or viral vector that contains at least one promoteroperably linked to a sequence encoding a regulatory RNA such as a siRNA,shRNA, miRNA, antisense, or a downstream gene or coding region or othernucleic acid sequence to be transcribed {e.g., a cDNA or genomic DNAfragment that encodes a protein, optionally, operably linked to sequencelying outside a coding region, or a sense and/or an antisense RNA codingregion, and/or RNA sequences lying outside a coding region). Thesequence(s) to be transcribed may include any target nucleic acidsequence whose expression is desired to be modulated. Transfection ortransformation of the expression vector into a recipient cell allows thecell to express RNA encoded by the expression vector. An expressionvector may be a genetically engineered plasmid, viral vector includingbut not limited to AAV, adenovirus, poxvirus, herpesvirus, retrovirus,lentevirus, and alphavirus, or artificial chromosome derived from, forexample, a bacteriophage, adenovirus, adeno-associated virus,retrovirus, poxvirus, or herpesvirus. Preferred for expression of dsRNAeffector molecules in the methods of the invention are RNA polymeraseIII Type 3 or “U6-type” RNA polymerase III promoters and multiple RNApolymerase III promoter expression constructs as taught in WO 06/033756.RNA polymerase II promoters including mammalian viral promoters, andmammalian including human cellular promoters may be utilized forexpression of longer RNAs including mRNAs. An expression construct canbe replicated in a living cell such as a bacterium or eukaryotic cell orit can be made synthetically. For purposes of this application, theterms “expression vector”, “expression construct”, “vector”, and“plasmid” are used interchangeably in the general illustrative sense andare not intended to limit the invention to a particular type ofexpression construct.

By “operably linked” is meant that a gene and one or moretranscriptional regulatory sequences, e.g., a promoter or enhancer, areconnected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequences.

By “promoter” is meant a minimal sequence sufficient to directtranscription of a gene. Also included in this definition are thosetranscription control elements (e.g., enhancers) that are sufficient torender promoter-dependent gene expression controllable in a celltype-specific, tissue-specific, or temporal-specific manner, or that areinducible by external signals or agents; such elements, which arewell-known to skilled artisans, may be found in a 5′ or 3′ region of agene or within an intron. Included are RNA pol I, RNA pol II and RNA polIII promoters, including RNA polymerase III Type 3 promoters such as HI,7SK, and U6. Polymerase III Type 3 promoters may advantageously be usedto express short oligonucleotides such as the shRNAs, siRNAs, and otheroligonucleotide RNA effector molecules of no more than 300 to 400nucleotides in length (see U.S. Pat. No. 5,624,802, Noonberg et al.). Apreferred RNA pol III 7SK promoter (7SK 4A) and expression constructscomprising multiple polymerase III promoters are taught in WO 06/033756.Also included are promoters that permit overexpression of mRNAs in thecytoplasm of the transfected cell, for instance to optimizeincorporation of expressed mRNA molecules into membrane exovesicles.

Expression plasmids that transcribe RNA effector molecules includingdsRNAs in either the cytoplasm or the nucleus may be utilized.Expression vectors may be designed to integrate into the chromosome oftransfected cells, for instance by homologous recombination.Alternatively, expression vectors may replicate in transfected cellsextrachromosomally. Nuclear transcription vectors for protein expressionare preferably designed to express polyadenylated 5′ capped RNA (forexample, a vector containing an RNA polymerase II promoter and a poly Asite) to facilitate export from the nucleus. Intracellular transcriptionmay also utilize bacteriophage T7 and SP6 promoters, i.e., bytransfecting a vector that coexpresses the appropriate RNA polymerasegene, which may be designed to transcribe in the cytoplasm or in thenucleus. Promoters for viral RNA polymerases, either DNA and RNAdependent, may also be used. Alternatively, dsRNA replicatingpolymerases can be used. Promoters for cellular polymerases such as RNAPolymerase I, II, or III or mitochondrial RNA polymerase may also beutilized. Tissue- or cell-specific promoters may be used to limitexpression of the dsRNA to the first transfected cell. Polymerase IIIpromoters are especially desirable for expression of small engineeredRNAs. See WO 06/033756, Multiple Polymerase III Promoter ExpressionConstructs, incorporated herein by reference. Preferred are polymeraseIII type 3 promoters, including various mammalian U6, HI, and 7SKpromoters, including the modified 7SK 4A promoter sequence taughttherein. See also, e.g., US 2005/0130184 Al, Xu et al., directed tomodified polymerase III promoters which utilize polymerase II enhancerelements, as well as US 2005/0130919 Al, Xu et al., directed toregulatable polymerase III and polymerase II promoters, the teaching ofwhich is hereby incorporated by reference. In some embodiments it may bedesirable to include one or more polymerase I, and/or one or morepolymerase II, and/or one or more polymerase III promoters in a singleexpression construct, as e.g., where it is desirable to utilize pol IIIpromoters to express one or more RNA effector molecules such as dsRNAsand one or more pol II promoters to express one or more targetingligands.

A desirable approach for cytoplasmic expression is to use endogenouspolymerases such as the mitochondrial RNA polymerase to make dsRNA inthe cytoplasm. These vectors are formed by designing DNA expressionconstructs that contain mitochondrial promoters upstream of the sequenceencoding the dsRNA. As described above for nuclear transcriptionvectors, dsRNA can be generated using two such promoters placed oneither side of the target sequence, such that the direction oftranscription from each promoter is opposing each other. Alternatively,two plasmids can be cotransfected. One of the plasmids is designed totranscribe one strand of the target sequence while the other is designedto transcribe the other strand. Single promoter constructs may bedeveloped such that two units of the target sequence are transcribed intandem, such that the second unit is in the reverse orientation withrespect to the other. Alternate strategies include the use of fillersequences between the tandem target sequences.

Cytoplasmic expression of dsRNA may also be achieved by transcription ofa single stranded RNA template in the nucleus of the transfected cell,which is then transported into the cytoplasm where it serves as atemplate for the transcription of dsRNA molecules, utilizing a singlesubgenomic promoter opposite in orientation with respect to the nuclearpromoter. The nuclear promoter generates one RNA strand that istransported into the cytoplasm, and the singular subgenomic promoter atthe 3′ end of the transcript is sufficient to generate its antisensecopy by an RNA dependent RNA polymerase to result in a cytoplasmic dsRNAspecies. Both cytoplasmic and nuclear transcription vectors may containa reporter gene to enable monitoring of cells that have taken up theplasmid. Any type of vector may be used, including plasmids, viralvectors, retroviral vectors, adenoviral vectors, AAV vectors, etc. Theuse of expression vectors to express double stranded RNA is alsodiscussed in detail in US 20040152117, which is herein incorporated byreference in its entirety.

If desired, inducible and repressible transcription systems can be usedto control the timing of the synthesis of RNA effector moleculesincluding mRNAs. Inducible and repressible regulatory systems involvethe use of promoter elements that contain sequences that bindprokaryotic or eukaryotic transcription factors upstream of the sequenceencoding dsRNA. In addition, these factors also carry protein domainsthat transactivate or transrepress the RNA polymerase II. The regulatorysystem also has the ability to bind a small molecule (e.g., a coinduceror a corepressor). The binding of the small molecule to the regulatoryprotein molecule (e.g., a transcription factor) results in eitherincreased or decreased affinity for the sequence element. Both inducibleand repressible systems can be developed using any of theinducer/transcription factor combinations by positioning the bindingsite appropriately with respect to the promoter sequence. Examples ofpreviously described inducible/repressible systems include lac, ara,Steroid-RU486, and ecdysone—Rheogene, Lac (Cronin et al. Genes &Development 15: 1506-1517, 2001), ara (Khlebnikov et al., J. Bacterid.2000 December; 182(24):7029-34), ecdysone (Rheogene, www.rheogene.com),RU48 (steroid, Wang X J, Liefer K M, Tsai S, O'Malley B W, Roop D R.,Proc Natl Acad Sci USA. 1999 Jul. 20; 96(15):8483-8), tet promoter(Rendal et al., Hum Gene Ther. 2002 January; 13(2):335-42. and Lamartinaet al., Hum Gene Ther. 2002 January; 13(2):199-210), or a promoterdisclosed in WO 00/63364, filed Apr. 19, 2000.

In one embodiment, among others, the present invention encompassesmethods of delivering nucleic acids to distal organs or tissues viaintramuscular, intradermal or subcutaneous administration andtransfection of muscle or skin cells with at least one nucleic acid invivo. When transfecting muscle cells, the expression construct comprisespolynucleotide sequences encoding the double stranded RNA that areoperably linked to regulatory elements operable in the muscle cell. Whentransfecting skin cells, the expression construct comprisespolynucleotide sequences encoding the double stranded RNA that areoperably linked to regulatory elements operable in the skin cell.Promoters and other regulatory elements operable in muscle and/or skincells are known in the art, and for instance include, but are notlimited to polymerase I, polymerase II, and polymerase III promoters(e.g., preferably pol III type 3 promoters including human and othermammalian U6, 7SK, and HI promoters) as well as mitochondrial promoters,e.g., human and other mammalian mitochondrial heavy and light chainpromoters. Short RNAs (fewer than 300-400 nt engineered RNAs such asshRNAs) are best expressed by pol I and/or pol III promoters. LongerRNAs, including mRNAs, are best expressed by pol II promoters, includingviral promoters such as CMV IEP, RSV LTR, SV40, etc. Bacteriophagepromoters such as T7, T3, SP6, etc. may also be utilized if the cell isalso provided with the cognate T7, T3, SP6 polymerase(s). For expressionin muscle and/or skin cells, such polymerase III promoters can be usedto express small dsRNAs and polymerase II promoters such as CMV(cytomegalovirus immediate early promoter) including HCMV (human), MCMV(murine), SCMV (simian), SV40, RSV, vaccinia, and other viral promoters;eukaryotic including mammalian polymerase II promoters such as theB-actin promoter, can be used to express a translatable mRNA encoding acell-surface protein, receptor, or targeting ligand or other desiredprotein. The mRNA may be translated in the first cell and/or a distalcell to which it is delivered.

Muscle cells include mammalian striated or skeletal myocytes, includingdifferentiated myocytes as well as undifferentiated myoblasts. Cardiacmuscle is also striated muscle. A myoblast is a type of stem cell thatexists in muscles. Skeletal muscle cells are called muscle fibers ormyocytes and are produced when myoblasts fuse together. Therefore,muscle fibers may have multiple nuclei. Myoblasts that do not formmuscle fibers differentiate into satellite cells. These satellite cellsremain adjacent to a muscle fiber, separated only by its cell membraneand by the endomycium (the connective tissue of collagen surrounding themuscle fiber).

“Intramuscular” administration means that the nucleic acid isadministered to muscle tissue and any muscle cell in the animal,including but not limited to skeletal muscle such as the deltoid, vastuslateralis, ventrogluteal, tibialis and dorsogluteal muscles. The meansby which intramuscular administration may be achieved includes needleinjection, needleless injection, electroporation, biolistic approaches,and any other method that can accomplish delivery into a muscle cell ora cell in muscle tissue. Such delivery may be directly into the muscletissue or via intravascular delivery into muscle cells or into cells inmuscle tissue supplied by such blood vessel(s). In the methods of theinvention comprising intramuscular administration, the RNAi agent orexpressed dsRNA or other nucleic acid-based therapeutic is designed toinhibit the function of a target polynucleotide in a cell of the mammalwhich is not a muscle cell.

In one aspect of the invention, such an expression construct encodingsequences homologous and complementary to one or more targetpolynucleotide sequences present in a liver cell is introduced into askeletal or other muscle cell and the function of one or more targetpolynucleotides in a liver hepatocyte is inhibited, e.g.,polynucleotides of a liver pathogen such as a hepatitis virus, includingHBV and/or HCV and HDV and HAV. In another aspect, the inventioninvolves introducing into an appropriate cell such as a muscle cell anexpression construct encoding sequences targeting genes in the liverresponsible for metabolic diseases or disorders such as high cholesterollevels for example, including but not limited to apolipoprotein B andpcsk9. Another such potential liver target is the genetic disorder alpha1-antitrypsin deficiency (αI-antitryspin deficiency, AIAD or Alpha-1),caused by a mutation which results in defective production of alpha1-antitrypsin (AIAT), leading to decreased AIAT activity in the bloodand lungs, and deposition of excessive abnormal AIAT protein in livercells. Treatment utilizing the methods of the invention would includeproviding an inhibitory dsRNA which targets the mutated AIAT sequence,while co-expressing an mRNA encoding the functional AIAT. This could beaccomplished by providing to muscle cells an expression vector(s)co-expressing the inhibitory dsRNA and an mRNA encoding the functionalprotein, both of which are delivered to liver cells.

“Intradermal” administration means in or into the skin, and can includeadministration to any layer of the dermis or epidermis, and deliveryinto any cell thereof. “Subcutaneous” administration means just underthe skin, or to the subcutaneous layer of the skin, and delivery intoany cell thereof. Also encompassed are epicutaneous forms ofadministration, i.e., with “epicutaneous” meaning on the surface of theskin (for instance using a transdermal patch, ointment, lotion or anyother suitable means). Skin cells include cells of the epidermis,including for example basal cells, melanocytes, Langerhans' cells,Merkel cells, sensory nerves, keratinocytes, and any other cell found inthe various layers of the epidermis including the basal layer, thesquamous cell layer, the stratum granulosum, the stratum lucidum and thestratum corneum. Skin cells also include cells of the dermis, includingvascular cells, lymph cells, cells of sweat and sebaceous glands, nervecells, fibroblasts, and any other cell found in the various layers ofthe dermis including the papillary layer and the reticular layer. Skincells also include those of the subcutis, i.e., the innermost layer ofthe skin, which includes fat and collagen cells.

The present invention encompasses delivery regimens where dsRNAs ornucleic acid vectors expressing the same are delivered to both skincells and muscle cells simultaneously or sequentially. The means bywhich intradermal, subcutaneous, and/or intramuscular administration maybe achieved includes needle injection, needleless injection,electroporation, biolistic approaches, and any other method that canaccomplish delivery into a muscle or skin cell or a cell in muscle orskin tissue.

In one embodiment, the present invention encompasses methods ofdelivering nucleic acids, including double stranded RNA molecules and/orpolynucleotide expression constructs encoding RNA molecules, e.g.,mRNAs, antisense, ribozyme or dsRNA including RNAi agents such as siRNA,shRNA, or miRNA, to a target cell in vitro or ex vivo by delivering anucleic acid to or expressing a nucleic acid in a cell that is competentfor distal cell targeting. Competent donor cells may also be utilizedfor production in cell culture of RNA-containing exovesicles, or forautologous or heterologous transplant into a recipient mammalianorganism. The cell may be a muscle cell, skin cell, stem cell, or anysuitable competent cell. A cell which is competent for distal celltargeting may be obtained e.g., from a potential mammalian recipient,e.g., a human recipient, or from another donor, transfected in vitro orex vivo with a selected polynucleotide expression construct or withselected RNA molecules, and implanted, e.g., subcutaneously orintramuscularly, into a recipient mammalian organism. The transplantedcell(s) may be autologous or heterologous with respect to the recipient.The recipient mammal may be an immunocompromised mammal or a mammaladministered immunosuppressants. The implanted cells may then serve as a“factory” for in vivo production of RNA effector molecules, e.g., RNAiand/or mRNA molecules, for transport to a distal cell. Such cells mayalso express targeting ligands or other proteins enhancing exovesicleproduction, transport, targeting, and/or uptake, such as the receptorfor HIV. The distal cell may be a liver cell such as a hepatocyte oranother cell. The RNA effector molecule may repress a target gene in theliver, e.g., a gene of a pathogen such as a hepatitis virus or anendogenous disease-related gene found in the liver. The distal targetcell may also be a non-liver cell, including any of the target cellsdescribed herein.

In one aspect, the invention relates to a method of dsRNA mediated geneinhibition or RNAi comprising delivering to muscle or skin cells of amammal in vivo a nucleic acid expression vector encoding an RNAi ordsRNA agent. As described above, the RNAi agent(s) may be one or morehairpin or duplex dsRNA molecules, including one or more short hairpindsRNA agents. The dsRNA expression vector may be DNA or RNA, includingplasmid DNA. The expression vector may be a viral vector such as AAV forexample. The polynucleotide expression vector may be supplied to themuscle as “naked” DNA or RNA (free from association with a transfectionfacilitating agent) or in association with an agent which facilitatestransfection or transfer into mammalian skin cells or muscle cells ormyocytes, including differentiated myocytes as well as undifferentiatedmyoblasts. In another aspect, the invention relates to such an RNAimethod involving delivery by intramuscular electroporation-mediatedtransfection of skeletal muscle or myocytes, or skin cells of a mammalin vivo with a vector or construct expressing dsRNA molecules.

Numerous agents may be used to facilitate transfection of mammalianmuscle cells in vivo including but not limited to polymer or peptidecomplexes, cationic amphiphiles, cationic lipids, cationic liposomicformulations, including the amino amide local anesthetic bupivacaine,particulates including gold particles utilized for biolistic or “genegun” delivery, polycationic or cationic amphiphile agents includingspermine and/or spermidine derivatives including various cholesterylspermine compounds. The polynucleotide expression vector is frequentlydelivered to the mammalian muscle or skin tissue formulated inassociation with or as a complex with one or more of suchtransfection-facilitating agents.

Bupivacaine is one of the amphiphilic amino amide local anestheticagents known to act as a transfection-facilitating agent for deliveryinto tissues including skeletal muscle of polynucleotide, includingplasmid DNA, encoding immunogenic proteins, e.g., antigens of pathogens,for use in the field of DNA vaccines. The proteins are expressed in themuscle cells, triggering both humoral and cellular immune responses. Seee.g., U.S. Pat. Nos. 6,217,900 and 6,383,512, “Vesicular Complexes andMethods of Making and Using the Same.” Methods of injecting “naked” DNAencoding immunogenic and other biologically active polypeptides intomuscle or skin are also known, see e.g., U.S. Pat. Nos. 5,580,859;5,589,466. U.S. Pat. No. 6,413,942 describes delivering into musclecells “naked” DNA encoding a secretable therapeutic polypeptide, e.g.,growth hormone, which is released into the circulation to achieve atherapeutic effect. Among the numerous polycationic or cationicamphiphile compounds useful for facilitating transfection ofpolynucleotides in vivo in mammalian cells are various cholesterylspermine or cholesteryl spermidine compounds including cholesterylspermine carbamates and combinations thereof used as taught e.g. in U.S.Pat. Nos. 5,837,533; 6,127,170; 6,379,965; 5,650,096; and 5,783,565; andUS 2006/0084617, published 20 Apr. 2006. These are just illustrative ofthe many chemically diverse agents known to those of skill in the artwhich may be employed to facilitate transfection of nucleic acidsincluding dsRNA expression constructs into mammalian muscle or skincells in accordance with the teaching of the invention.

Such naked and complexed nucleic acids may be used in delivery methodsincluding needle and/or needleless injection directly into skin and/ormuscle tissue, e.g., DNA expression vector complexed with 0.25%bupivacaine in a suitable vehicle for injection as taught above, as wellas delivery to muscle or skin cells by the vascular route, such as thehydrodynamic method whereby increased intravascular pressure producesincreased vascular permeability to the passage of molecules includingnucleic acids into the interstitial space of muscle and skin tissue andincreased uptake of such molecules by cells of the skin and muscle. Suchincreased intravascular pressure may be achieved through a combinationof externally applied pressure e.g. tourniquet or cuff; and/or increasedvolume of drug administration; and/or increased speed of administration.Volumes administered to the limb of a mammal can be 250 ml, 500 ml, upto a liter or more. In one aspect, the nucleic acid is a DNA plasmidexpression vector which can be administered in relatively large doseswithout toxicity, e.g. 100 mg, 200 mg, 350 mg, 500 mg to 1, 2, or 5grams or more. Thus e.g. a delivery vehicle may be formulated comprising350-500 mg naked or complexed DNA expression vector in 100 to 500 ml(e.g. 2 mg/ml) of citrate buffered 5% dextrose in water for injection(D5W). When administered rapidly into the afferent or efferent vascularsystem supplying a mammalian limb subjected to externally appliedpressure, such a formulation provides a large amount of agent in theinterstitial space of muscle cells, thereby facilitating transfection ofcells by mass action. The formulation may be hypotonic, isotonic, orhypertonic. Administration of a hypertonic delivery vehicle may increasethe transport of molecules such as DNA or other nucleic acids intoadjacent muscle cells. See additional discussion below. Other deliveryvehicles, excipients, and methods suitable for use in the methods of theinvention may be formulated by those of skill in the art ofpharmaceutical sciences, see e.g., the teaching of Remington'sPharmaceutical Sciences 18^(th) Ed. (1990); Remington: The Science andPractice of Pharmacy, 20^(th) Ed. (2000), 21^(st) Ed. (2005). Theteaching of all these cited references is incorporated herein byreference.

In another aspect, nucleic acids including dsRNA expression constructsare delivered into mammalian skin cells or striated or skeletal myocytesand/or myoblasts in vivo through electroporation. See, e.g., theformulations and methodology of electroporation of nucleic acidconstructs into mammalian muscle cells as taught in US 2004/0014645 Al“Increased delivery of a nucleic acid construct in vivo by thepoly-L-glutamate (‘PLG’) system” and the methods and devices forelectroporation taught in e.g., US 2005/0052630A1 “Constant currentelectroporation device and methods of use.” See also US 2005/0070841 Aland US 2004/0059285 Al “Electroporation device and injection apparatus”and US 2004/0092907 Al “Method for muscle delivery of drugs, nucleicacids and other compounds.” The various parameters including electricfield strength required for electroporation of any known cell typeincluding muscle and skin are generally known in the relevant researchliterature as well as numerous patents and applications in the field.See e.g., U.S. Pat. No. 6,678,556 “Electrical field therapy with reducedhistopathological change in muscle”; U.S. Pat. No. 7,171,264“Intradermal delivery of active agents by needle-free injection andelectroporation”; and U.S. Pat. No. 7,173,116, which teachesformulations for gene delivery via electroporation, includingformulations of various anionic polymers including poly-L-glutamate.Apparatus for therapeutic application of electroporation are availablecommercially, e.g., the MedPulser® DNA Electroporation Therapy System(Inovio/Genetronics, San Diego, Calif.), and are described in patentssuch as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No.5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S.Pat. No. 6,233,482; electroporation may also be used for transfection ofcells in vitro as described e.g. in US20070128708A1 Electroporation invivo into mammalian muscle cells presents an attractive alternative forexperimental applications as well as a promising delivery method fortherapeutic applications in mammals including humans, with clinicaltrials underway in the DNA vaccines field. Electroporation may also beutilized to deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into muscle and skin cells ofnucleic acids including expression constructs utilizing any of the manyavailable devices and electroporation systems known to those of skill inthe art presents an exciting new means for delivering an RNA of interestto a distal target cell, tissue, or organ such as a hepatocyte or otherliver cell.

In another embodiment, administration may be via needle injection,needleless injection, e.g., the Biojector® 2000 needleless injectiondevice (Bioject Needle-free Injection Systems, Tualatin, Oreg.), whichcan be adjusted to deliver a liquid medication to various depthsincluding intradermal, subcutaneous, and/or intramuscular. Othercommercially available needleless injection systems include Dermo-jet(Robbins Instruments, Chatham, N.J.). Needleless injection relies on ahigh-pressure stream of the medication itself to penetrate the skin. Asthe fluid stream forces its way through the tissue, it follows the pathof least resistance, resulting in a widely dispersed, spiderweb-likedistribution of the medication. A transdermal patch might also beemployed, as well as a biolistic injector or “gene gun” device capableof shooting a plasmid expression vector into cells, including cells inskin, the subcutaneous region, and/or muscle tissue. Biolistic or genegun devices for in vivo delivery to mammals are commercially available,as e.g., the Helios Gene Gun (Bio-Rad, Hercules, Calif.) See, e.g., U.S.Pat. Nos. 5,830,877 and 6,723,077, which are herein incorporated byreference in their entireties.

In another aspect, intramuscular administration may be achieved throughhydrodynamic intravascular methods which utilize various means, e.g.,increased pressure, to promote delivery to cells including muscle, e.g.,the Mirus Pathway IVT™ Gene Delivery methods (Mirus, Madison, Wis.)which utilize a cuff or tourniquet to restrict blood flow and increasepressure within the vessel in order to facilitate intravascular deliveryof nucleic acids such as plasmid expression vectors to limb muscle. Anyof a variety of methods known in the art may be used to increase thepassage of a nucleic acid from afferent or efferent blood vessels intocells, including parenchymal cells of adjacent tissues. For example,increased vessel permeability to nucleic acids and other molecules maybe achieved by the external application of pressure by a cuff such as ablood pressure cuff or tourniquet at a location distal to the site ofnucleic acid administration and/or through increased intravascularpressure achieved by administering a selected pharmaceutical formulationin a relatively large injection volume and/or through rapid deliveryand/or administration of biologically active agents such as papaverine,hyaluronidase, etc. The specific parameters for achieving optimalincreases in vascular permeability in test subjects such as laboratoryanimals as well as human subjects are well within the level of skill inthe arts of animal science, anatomy, physiology, pharmacology andclinical medicine. For example, optimal injection volume is related tothe size of the animal to be injected as well as target tissue volume,e.g., volumes of 0.03 ml/g to 0.1 ml/g of body weight or greater may berequired, with injection volumes of 70 to 200 ml reported for primates.Delivery to skeletal muscle tissue of a limb may be achieved by rapidintravascular injection or delivery through needle, catheter etc. of arelatively large volume (e.g., >5 ml per rat limb or >70 ml for aprimate) with concomitant external application of pressure e.g. with acuff or tourniquet, such that pressure within the vessel is increasedand permeability to outward movement of oligonucleotide, polynucleotide,etc. is enhanced. See e.g., US 2004/0259828, U.S. Pat. No. 6,379,966; US2007/0244067; Hagstrom et al., Molecular Therapy (2004) Vol. 10 (No. 2),386-398, Herweijer and Wolff, Gene Therapy (2007) 14, 99-107; Lewis andWolff, Advanced Drug Delivery Reviews 59 (2007) 115-123; the teaching ofeach of which is incorporated herein by reference. Such methods may beemployed to deliver nucleic acids, including RNAs, DNAs, and mixturesthereof, to various parenchymal cells of tissues in a mammal, includingcells of striated muscle as e.g., myoblasts, satellite cells, myotubulesand myofibers, by delivery into an afferent or efferent blood vesselsupplying the tissue, preferably a vessel of the arterial system such asan artery, arteriole, sinusoids, and/or capillary, but in someembodiments a vessel of the venous system, including a vein, venule andcapillary. Increased transfection of such cells, including muscle cells,may be achieved e.g. by increasing the permeability of an afferentvessel proximal to the target tissue into which a selected nucleic acidis administered as taught e.g. in U.S. Pat. No. 7,148,205,“Intravascular delivery of non-viral nucleic acid”. The nucleic acid,e.g., an RNA or a DNA expression vector, may be “naked” (i.e., free fromagents which associate or complex with the nucleic acid and promotetransfection) or associated or complexed with any one or more agentsincluding amphipathic or amphiphilic compounds such as cationicamphiphiles, cholesterol and/or spermine containing complexes asdescribed elsewhere herein. Various means may be used in order toincrease vessel permeability, e.g., the naked or complexed nucleic acidmay be supplied in a relatively large solution volume, and/or withphysical measures to increase the pressure within the vessel by applyingdistal pressure and/or decreasing the vessel lumen, as e.g., with atourniquet or cuff.

While some embodiments encompass transfecting skin cells or skeletalmuscle cells or other competent cells in an animal with a nucleic acidencoding a dsRNA, methods wherein dsRNA is directly transfected intoskin or muscle cells or other competent targeting cells are alsoencompassed. For instance, the invention includes a method of deliveringat least one dsRNA to a target organ or tissue in an animal comprisingtransfecting skeletal skin or muscle cells or other competent targetingcells in said animal with said dsRNA, wherein said transfection resultsin said dsRNA being delivered to said other target organ or tissue.

Suitable dsRNA molecules for delivery to skin and muscle cells and othercompetent targeting cells include shRNAs and siRNAs for RNAi embodimentsand are typically between about 15 to about 50 base pairs, and moreparticularly between about 19 and about 29 base pairs. The dsRNA andcomplexes or other formulations containing the same may be delivered toskin or muscle cells or other cells using any method known in the art,including those discussed above with regard to expression vectors.

Some dsRNA sequences, possibly in certain cell types and through certaindelivery methods, may result in an interferon response. The methods ofthe invention may be performed so as not to trigger an interferon/PKRresponse, for instance by using shorter dsRNA molecules between 20 to 25base pairs, by expressing dsRNA molecules intracellularly, or by usingother methods known in the art. See US Application 200401521 17, whichis herein incorporated by reference. For instance, one of the componentsof an interferon response is the induction of the interferon-inducedprotein kinase PKR. To prevent an interferon response, interferon andPKR responses may be silenced in the transfected and target cells usinga dsRNA species directed against the mRNAs that encode proteins involvedin the response. Alternatively, interferon response promoters aresilenced using dsRNA, or the expression of proteins or transcriptionfactors that bind interferon response element (IRE) sequences isabolished using dsRNA or other known techniques.

By “under conditions that inhibit or prevent an interferon response or adsRNA stress response” is meant conditions that prevent or inhibit oneor more interferon responses or cellular RNA stress responses involvingcell toxicity, cell death, an anti-proliferative response, or adecreased ability of a dsRNA to carry out a PTGS event. These responsesinclude, but are not limited to, interferon induction (both Type 1 andType II), induction of one or more interferon stimulated genes, PKRactivation, 2′5′-OAS activation, and any downstream cellular and/ororganismal sequelae that result from the activation/induction of one ormore of these responses. By “organismal sequelae” is meant any effect(s)in a whole animal, organ, or more locally (e.g., at a site of injection)caused by the stress response. Exemplary manifestations include elevatedcytokine production, local inflammation, and necrosis. Desirably theconditions that inhibit these responses are such that not more thanabout 95%, 90%, 80%, 75%, 60%, 40%, or 25%, and most desirably not morethan about 10% of the cells undergo cell toxicity, cell death, or adecreased ability to carry out a PTGS event, compared to a cell notexposed to such interferon response inhibiting conditions, all otherconditions being equal (e.g., same cell type, same transformation withthe same dsRNA).

Apoptosis, interferon induction, 2′5′ OAS activation/induction, PKRinduction/activation, anti-proliferative responses, and cytopathiceffects are all indicators for the RNA stress response pathway.Exemplary assays that can be used to measure the induction of an RNAstress response as described herein include a TUNEL assay to detectapoptotic cells, ELISA assays to detect the induction of alpha, beta andgamma interferon, ribosomal RNA fragmentation analysis to detectactivation of 2′5′ OAS, measurement of phosphorylated eIF2a as anindicator of PKR (protein kinase RNA inducible) activation,proliferation assays to detect changes in cellular proliferation, andmicroscopic analysis of cells to identify cellular cytopathic effects.See, e.g., US Application 20040152117, which is herein incorporated byreference.

As noted above, while not wishing to be bound by any particularmechanism, the present inventors have hypothesized that double-strandedRNA molecules transcribed from expression constructs in muscle and skincells are delivered to the liver, possibly inside exovesicles formedfrom membrane blebbing or shedding at the surface of the transfectedcell. In line with this hypothesis, the present invention also includesco-transfecting or co-expressing, along with an RNA of interestincluding dsRNA or therapeutic mRNA in muscle cells or other competentcells, genes encoding surface or transmembrane ligands specific for thetarget cell, organ or tissue of interest. Genes encoding targetingligands may be expressed on the same vector as the dsRNA, for instancefrom a separate promoter, or may be expressed from a separate vector. Insome embodiments, among others, the dsRNA is expressed from a polIIIpromoter and the targeting ligand is expressed from a polII promoter.Incorporation of such surface ligands into exovesicles could furtherenhance delivery to the liver, or facilitate delivery to other targetssuch as cancer cells or immune cells.

For instance, the present invention encompasses methods whereby skin ormuscle cells or other competent targeting cells are transfected with (1)eiRNA or dsRNA or dsRNA complexes and (2) an expression vector encodinga cell-surface ligand that specifically binds to a receptor on thetarget cell. The eiRNA expression vector and the ligand-encodingexpression vector may be a single expression vector or two differentexpression vectors. As an example, expressing a viral glycoprotein (suchas the HIV envelope glycoprotein gp120) in muscle or skin cells inaddition to HIV-targeting dsRNAs should lead to the formation ofanti-HIV dsRNA-containing muscle exovesicles comprising HIV glycoproteinon the surface. These exovesicles now have the potential to be taken upby the T cells and other CD4+ immunocytes that HIV infects. Othersuitable cell surface ligands and target cells include the influenza Ahemaglutinin (HA) receptor binding domain which recognizes and interactswith an oligosaccharide on the surface of respiratory epithelial cells.Since avian influenza A viruses and human influenza A virusespreferentially target different epithelial cell-surface oligosaccharidereceptors (e.g., epithelial cell receptors identified as glycansterminated by an <<2,3-linked sialic acid (SA) that preferentially bindavian strains and glycans terminated by an ft2,6-linked SA that bindhuman strains. J. Virol., August 2006, p. 7469-7480, Vol. 80, No. 15),expression constructs can be designed to express dsRNAs active againsthuman and/or avian influenza A viruses as well as influenza A receptorbinding domains that preferentially target the human receptor and/or theavian receptor. Still other examples of cell surface ligands and targetcells will be readily apparent to those of skill in the art of virology.For example, HBsAg can be encoded to further facilitate delivery tohepatocytes. Any viral receptor binding protein encoded by a virus canbe expressed to target the blebs or exovesicles to cells infected by orcapable of being infected by the respective viruses. Cells can betargeted prophylactically or therapeutically.

Any suitable cell surface ligand having specificity for a target cellsurface receptor may be expressed in transfected cells, by cloning thegene for the cell surface ligand behind a promoter operable in thetransfected cell such that the cell surface ligand is expressed anddisplayed on the surface of the transfected cell. In order topreferentially localize expressed cell surface ligands into exovesicles,the gene for the cell surface ligand may be fused to or be engineered toincorporate suitable targeting signals from proteins known to beincorporated into exovesicles at the surface of the transfected cell.

For example, as discussed above, galectin is one protein expressed athigh levels in skeletal and smooth muscle cells, which has been observedto become specifically concentrated in plasma membrane evaginations andextruded in extracellular vesicles. Cooper and Barondes, 1990; Cooper,1997; Harrison and Wilson, 1992. A variety of other galectin genes havebeen detected in different cell types, each lacking a signal sequenceand containing a highly conserved core sequence of about 130 to 135amino acids containing a carbohydrate recognition domain (CRD) betweenabout residues 30 and 90. See Cooper and Barondes, 1999. Walter Nickeland colleagues have found that the CRD domain in galectin is necessaryfor export, suggesting that the galectin export machinery makes use ofβ-galactosidase-containing surface molecules as export receptors forintracellular galectin-1. Seelenmeyer et al., 2005. Accordingly, it maybe possible to target other cell surface ligands to exovesicleevaginations in the cell membrane by fusing the coding regions for suchcell surface ligands in frame to all or part of the galectin CRD domaincoding region. The complete amino acid sequence of galectin-1 has beendetermined from human, as well as several other species including cow,rat, mouse, chicken and electric eel. Inagaki et al., 2000; Hirabayashiet al., 1988, Complete amino acid sequence of a β-galactosidase-bindinglectin from human placenta, J. Biochem. (Tokyo) 104:1-4; Abbott andFeizi, 1989, Evidence that the 14 kDa soluble beta-galactosidase-bindinglectin in man is encoded by a single gene, Biochem. J. 259: 291-94;Couraud et al., 1989, Molecular cloning, characterization, andexpression of a human 14 kDa lectin, J. Biol. Chem. 264: 1310-16.

Galectin-3 is another member of the galectin family shown to be secretedby the pinching off of evaginating membrane domains and the release ofextracellular vesicles where it is protected from proteolysis. Krzeslakand Lipinska, 2004. Electron microscopy has demonstrated that thevesicles are morphologically heterogenous and have a small size (up toabout 0.5 microns). Galectin-3 is unique in the galectin family inpossessing an extra N-terminal domain consisting of 100-150 amino acidsdepending on the species of origin, which has been proposed to containtargeting information for non-classical secretion. Further, at least onegroup has shown that the addition of this N-terminal segment to anormally cytosolic protein such as chloramphenicol acetyltransferase(CAT) resulted in efficient export of the fusion protein fromtransfected Cos cells. See Mehul and Hughes, 1997. Accordingly, itshould be possible to target other cell surface ligands to exovesicleevaginations in the cell membrane by fusing the coding regions for suchcell surface ligands in frame to all or part of the coding region forthis galectin-3 N-terminal domain. Other classical methods of expressingtargeting ligands on the cell surface may also be used.

The invention also encompasses methods wherein genes for cell surfacereceptors having binding specificity for cell surface ligands expressedin the competent cell are cloned, transfected into and expressed intarget cells in vivo. Alternatively, recombinant genes may be introducedinto target cells that upregulate expression of targeted cell surfacereceptors. Administering certain drugs to the patient may also result inthe upregulation of target cell surface receptors depending on thetarget cell surface receptor of interest.

In some embodiments, the methods of the present invention may furthercomprise isolating at least one exovesicle from a transfected cell,wherein the isolated exovesicle contains the nucleic acid, for instancea dsRNA, and contacting said target cell with the isolated exovesicle.Exovesicle-producing cells may be transfected in vitro or in vivo, anddsRNA-containing exovesicles thereafter isolated and used to contacttarget cells. Methods for transfecting exovesicle-producing cells andcell lines in vitro with expression vectors and dsRNA compositions areknown in the art, as are methods of isolating exovesicles. For example,Mehul and Hughes have disclosed a method of isolating agalectin-3-enriched vesicular fraction from murine macrophages and Cos-7cells transfected with galectin-3 expressing plasmid. Mehul and Hughes,1997, Plasma membrane targeting, vesicular budding, and release ofgalectin 3 from the cytoplasm of mammalian cells during secretion, J.Cell Sci. 110: 1169-78.

The methods of the present invention may be used in methods of treatingand preventing diseases in mammals, particularly humans. Any mammal maybe treated by the methods of the present invention, including but notlimited to humans, primates, laboratory animals such as mice, rats,rabbits, guinea pigs, etc, farm animals such as cows, sheep, pigs,horses, goats, etc., as well as domestic animals including cats, dogs,etc. The experiments described herein show that diseases may be treatedtherapeutically or prophylactically. In particular, the presentinvention demonstrates that dsRNA expressed in or just below the skin,or in skeletal muscle may be delivered to other organs or tissues.Accordingly, the present invention may be used to treat or prevent anydisease of an organ or tissue in which a reduction in gene expression iseffective. For example, the invention includes a method of treating apatient suffering from an infection of an organ or tissue, for instancea viral infection, by delivering viral specific dsRNAs anddsRNA-containing viral drugs to the infected target cell.

In the case of hepatitis B and/or C viral infection, for example, HBV-and/or HCV-specific dsRNAs or other dsRNA-containing HBV- orHCV-specific drugs are delivered to the liver by intramuscular,intradermal or subcutaneous administration or by administration to anyother competent targeting cell. Any HBV and/or HCV gene sequence may betargeted. Other liver viral diseases treatable by the methods of thepresent invention include CMV-induced hepatitis and HAV, HBV and/or HDV,and HEV and HAV.

In the case of HIV infection, for example, HIV-specific dsRNAs or otherdsRNA-containing HIV specific drugs are delivered to T cells, T cellprogenitors or other cells susceptible to HIV infection byintramuscular, intradermal or subcutaneous administration or byadministration to any other competent targeting cell. Any suitable HIVgene sequence may be targeted, including but not limited to env (gp12),gag, and pol. As with any highly mutable virus, it is desirable todeliver a plurality of dsRNA molecules targeting two, three, four, fiveor more different HIV viral gene sequences and/or different HIV viralgenes, e.g., one or more sequences from HIV env (gp120 and gp 41), HIVgag (p24, pi 7, etc.), and/or HIV pol (including protease, p31integrase, pi 5 RNase, and p51 reverse transcriptase) which code forstructural proteins, and/or one or more sequences from the HIVregulatory genes tat, rev, nef, vif, vpr and vpu, which code forproteins that control the ability of HIV to infect a cell, produce newcopies of virus, or cause disease. In the case of HIV, as discussedabove, expressing HIV gp120 glycoprotein in muscle cells or othercompetent cells in addition to HIV-targeted dsRNA should lead to theformation of dsRNA-containing exovesicles comprising HIV glycoprotein onthe surface. These exovesicles now have the potential to be taken up bythe T cells and other CD4+ immunocytes that HIV infects. Any otherligand that binds to a receptor on the surface of T cells could also beused to target dsRNA or therapeutic mRNAs to T cells.

Other viruses that may be targeted by the present invention include, butare not limited to, influenza, RSV, rabies, picornarirus, polio,coxsacchie, herpes simplex virus Type I and 2, St. Louis encephalitis,Epstein-Barr, myxoviruses, JC, coxsakieviruses B, togaviruses, measles,paramyxoviruses, echoviruses, bunyaviruses, cytomegaloviruses,varicella-zoster, mumps, equine encephalitis, lymphocyticchoriomeningitis, rhabodoviruses including rabies, simian virus 40,human polyoma virus, parvoviruses, papilloma viruses, primateadenoviruses, coronavirases, retroviruses, Dengue, yellow fever,Japanese encephalitis virus, and/or BK, or any viruses of thespecies/family Astoviridae, Togaviridae, Flaviviridae, paramyxoviridae,arteriviruses, Rhabdoviridae, Filoviridae, orthomyxoviridae,bunyaviridae, arenaviridae, reoviridae, Birnaviridae, circoviridae,adenoviridae, Iridoviridae, Retrovirus, Herpesvirus, Hepadenovirus,Poxvirus, Parvovirus, Papillomavirus, and Papovavirus.

Prion RNA, e.g., mRNA, can also be targeted for repression.

Infections by other pathogens may also be treated or prevented using themethods of the present invention, including protozoa, bacteria, yeast,and fungal infections. For example, for intracellular parasites such asPlasmodia, pathogen-specific dsRNAs or other dsRNA-containing drugseffective against the pathogen may be delivered to cells susceptible toinfection by intramuscular, intradermal or subcutaneous administrationor by administration to any other competent targeting cell. The methodsof the present invention are especially useful for treating hepatocyteinfection by Plasmodia species by intramuscular, intradermal orsubcutaneous administration and/or expression and delivery ofPlasmodium-specific dsRNA to the liver.

As discussed above, the present inventors hypothesize that expresseddsRNAs in muscle and skin cells are delivered to distal organs ortissues after being incorporated into exovesicles at the transfectedcell surface. This incorporation could be a passive mechanism based onthe sheer numbers of expressed dsRNA molecules in the cell cytoplasm,where dsRNAs are captured as a result of a natural process and carriedalong in the vesicle. In this regard, as described above, the inventionwould encompass methods of delivering any nucleic acid or other cellularcomponent expressed at sufficient levels so as to be incorporated intomembrane exovesicles as they bleb from the cell surface, including mRNAsexpressed from therapeutic genes, i.e., gene therapy.

The following examples are provided to describe and illustrate thepresent invention. As such, they should not be construed to limit thescope of the invention. Those in the art will well appreciate that manyother embodiments also fall within the scope of the invention, as it isdescribed hereinabove and in the claims.

EXAMPLES

As reported herein the present inventors have found that intramusculardelivery of DNA/Bupivacaine complexes, or intramuscularelectroporation-mediated transfection of muscle cells with a vector orconstruct expressing dsRNA molecules, e.g, shRNA (RNAi) molecules, issurprisingly able to reduce the expression of target genes expressed inthe liver. Several sets of experiments are described which havedemonstrated gene silencing in mouse liver, mediated by an expressedinterfering RNA (eiRNA) expression vector injected into the muscle.Experimental designs differ most significantly in the sequence ofadministration of the test material (therapeutic vs. prophylactic), andthe end product that is being assayed to ascertain the level of genesilencing taking place. In the therapeutic model, the target molecule(plasmid expressing an RNA to be silenced) is administered before thedrug molecule (plasmid expressing the shRNA silencing molecule) isadministered. The reverse is true for the prophylactic model. In allexperiments, the target RNA to be silenced is measured indirectly byquantitating the level of protein or enzymatic activity which istranslated from the target RNA. In some experiments, the HBV surfaceantigen (sAg) is the measured end product. In others, luciferaseactivity is the measured end product.

In all the experiments described here, the hydrodynamic protocol usedfor injection of the target plasmid directs the plasmid to the liver,and is assayed at time points when the plasmid is expressed exclusivelyin the liver. In certain sets of experiments, expression of the targetRNA is further restricted to the liver because a liver-specific promoteris used, which does not permit significant levels of transcription inother tissues. Since the liver secretes protein products into thecirculatory system of the animal it is possible and convenient to samplethe blood serum of the animal to measure the liver-based expression ofthe target plasmid in some of these experiments. Since obtaining a serumsample from the animal does not involve sacrificing the animal orotherwise disturbing liver function, this allows the investigator torepeatedly sample the target RNA levels in liver at multiple timepoints, indirectly by measuring the protein product or enzymaticactivity of the indicator protein in the blood.

In all experiments “NUC050” refers to the eiRNA plasmid that isHBV-specific and which mediates sAg gene silencing. NUC049 is a negativecontrol eiRNA plasmid (containing a mutated version of an shRNA derivedfrom NUC050).

Example 1 Therapeutic Inhibition of HBV sAg Gene Expression In Vivo byIntramuscular Injection

In the therapeutic model, animals receive exogenous RNA target in theform of a plasmid expressing the surface antigen coding sequence of HBVvia hydrodynamic tail vein injection. Hydrodynamic injection is a methodusing a large volume with rapid injection time, to preferentially directthe DNA plasmid to the liver where it is expressed.

To obtain mice expressing a target gene in liver, HBsAg (surfaceantigen) cDNA was placed under the control of a liver-specific promoterin a commercially available plasmid vector (pLIVE). On Day −10, thisvector was injected hydrodynamically into an immunodeficient strain ofmice (NOD.CB17-Pkrdc^(scld)/J). In this way, DNA is largely localized toliver hepatocytes and the tissue-specific promoter further restrictsexpression of the sAg mRNA to these cells. Since the mice areimmunodeficient, they are able to express the target gene for longperiods of time (greater than a month) because immune response to theforeign protein encoded by the target gene will not be made.

Five days following target plasmid administration (Day −5), the micewere bled to determine levels of circulating HBsAg in serum.

On Day 0, mice were intramuscularly injected with the NUC050 vector at1.0 mg/ml in a 0.25% bupivacaine solution in a total volume of 50 ul.The NUC050 vector encodes four different shRNA molecules which targetvarious portions of the hepatitis B genome for degradation via thecellular RNAi mechanism. Three of these target the HBsAg regionscontained in the target vector preadministered to the mice. A controlgroup of mice was treated with the negative control NUC049 plasmid. (Insome experiments, mice received another negative control plasmid, pGL2,which expresses luciferase mRNA but no shRNA molecules.)

Animals were subsequently bled again on Days 4, 11, 19, and 26 days posteiRNA vector administration, respectively, and levels of serum HBsAgwere determined using an HBsAg ELISA commercially available (Bio-Rad,Hercules, Calif. HBsAg 3.0 EIA cat#. 32591). From 5 to 8 mice were usedfor each treatment group and the results are presented as HBsAg percentof initial HBsAg values (pre-treatment). The ability of the NUC050vector treatment to decrease HBsAg expression was estimated bycalculating, for each bleed day, a normalized difference value ofaverage HBsAg levels for control minus experimental groups.Normalization was done by calculating the percent of HBsAg for eachsubsequent bleed day, relative to the HBsAg values on pretreatment.Wilcoxon statistical tests were used to assess the significance of theresults. Experimental vs. control differences were taken to besignificant at p-values less than 0.05.

Day 11 and Day 19 show statistically significant difference averages of−36% and −28% respectively, between NUC050 and NUC049 (p=0.0088,p=0.0339). (These values reflect underlying normalized average values of56% and 93% prebleed (pretreatment) values for NUC050 and NUC049respectively. For Day 11 the normalized average values compared toPrebleed values were 54% (NUC050) vs. 82% (NUC049) for Day 19.). Averagevalues for all bleed days for NUC050 were substantially lower than allcorresponding NUC049 values, but reached statistical significance onDays 11 and 19. See FIG. 1.

Example 2 Therapeutic Inhibition of HBV sAg Gene Expression In Vivo byIntramuscular Injection

Mice (immunodeficient strain NOD.CB17-Pkrdc^(scld)/J) were given targetHBV sAg (surface antigen) expression plasmid (directed to liver byhydrodynamic injection as described in Example 1) several days beforebeing injected IM (intramuscularly) with 1) eiRNA vector expressing antiHBV shRNA, 2) negative control vector, or 3) left untreated after theinitial injection of target plasmid. The eiRNA vectors for IM injectionwere formulated with 0.25% bupivacaine. Expression levels of the sAg,produced in the liver, were monitored by sAg ELISA of serum samples overthe course of about 4 weeks.

In this model, sAg expression is expressed at a given level followinginjection, and then declines gradually over several weeks, if leftsimply to its own course, as seen in the mice left untreated after theinitial target plasmid injection. By administering the eiRNA anti-sAgshRNA plasmid, the rate of decline of sAg increases over time relativeto mice not treated after the injection of target HBsAg expressionplasmid, or relative to mice given a negative control plasmid instead ofthe anti HBV eiRNA. An overall lower level of expression of sAg can alsobe observed in experimental vs. control groups of mice. Success rate andefficiency of muscle transfection by injection may be demonstrated bycotransfecting a reporter gene (e.g., by encoding a reporter gene suchas EGFP on the eiRNA plasmids) and monitoring its expression either inwhole muscle or tissue sections after injection.

Example 3 Prophylactic Inhibition of HBV sAg Gene Expression In Vivo byIntramuscular Injection

Mice (immunocompetent strain C57B1/6) were given a single intramusculardose of the anti HBV shRNA effector plasmid, NUC050, or a negativecontrol plasmid, which was followed three days later by hydrodynamicinjection (via tail vein) of the target HBsAg plasmid (same targetplasmid as in Exp 1 above). The control plasmid used in this experimentwas the commercial vector pGL2, which encodes no protein and is used asan irrelevant plasmid control with respect to eiRNA activity. The IMinjections were formulated with bupivacaine (0.25% w/v) with a DNAconcentration of 1.0 mg/ml. 100 ul of the bupivacaine formulation wasadministered IM to each mouse (100 ug DNA).

Mice were bled for HBsAg ELISA assay at 3, 6, 9, and 13 days after thehydrodynamic administration of target plasmid.

Since the target plasmid encoding HBsAg mRNA and protein is givensubsequent to the effector plasmid in the prophylactic model it is notpossible to obtain a “baseline” sAg value for these mice prior to drugadministration. Hydrodynamic injection is known to produce generallyrobust but highly variable levels of sAg in this type of animal model.Therefore, the sAg level determined at any time point reflects anunknown variation in the potentially maximal level of sAg expressionfrom the target plasmid as well as the experimental variable ofinterest, which is the gene silencing effect of the eiRNA plasmid. Forthis reason, a comparison of the overall levels of sAg in the controland experimental groups is not conclusive, and a rate analysis (alsocalled “slope” analysis”) is performed.

The rate of decline of sAg levels between days 3 and 13 is approximatedfirst by fitting a line to the data curve, and then comparing the slopesof this best fit line between control and experimental groups. Theresults revealed an increase in the decay rate of sAg mediated by theNUC050 eiRNA plasmid, relative to negative control plasmid or mice givenonly the target plasmid. The rates of decrease range from 1.4 to 1.7times greater for eiRNA treatment compared to control mice.

Example 4 Prophylactic Model Using Immunodeficient Strain NOD. CBI7-Pkrdc^(scld)/J, Multiple Dosing and Luciferase Readout

In this experiment, using bupivacaine formulations only, of the NUC050(eiRNA against HBV sAg) and the NUC049 (negative control) vectors, adual reporter system was used to normalize for the liver-directedexpression of target plasmid.

The target plasmid contains two separate genes encoding luciferases fromtwo different organisms, firefly (FF) and jellyfish (Renilla or “JF”).See WO 04076629A3: Methods and constructs for evaluation of rnai targetsand effector molecules. Because each produces a different wavelength ofluminescence upon hydrolysis of luciferin substrate, the two signals canbe measured in the same sample. The renilla luciferase mRNA isengineered as a fusion mRNA with sequence elements present in the HBVgenome, such that the fusion mRNA becomes a target for the NUC050 eiRNAplasmid, and the signal from renilla is a measure of the gene silencingeffect of NUC050. Activity of NUC050 will therefore result in areduction of the renilla luciferase signal even though it targets HBVsequences because the HBV sequences are present at the end of therenilla mRNA. The FF luciferase signal serves as a normalizationstandard to correct for overall variability in target plasmiddelivery/transfection, because it is not subject to down modulation bythe eiRNA effector, but is dependent on the same plasmid for expression.Thus, taking the ratio, JF:FF luciferase normalizes for theliver-directed expression of plasmid. In cases where an insufficient FFluciferase is observed, it can be concluded that the plasmid was notdelivered, and therefore the sample is not valid for analysis.

In this experiment, 25 animals were used for each treatment group.Levels of FF luciferase were acceptable for 19/25 animals in the eiRNA(NUC050) group and for 17/25 animals in the control (NUC049) group. Abox-and-whisker plot diagram of all ratio values indicates a clear shiftto lower renilla:FF ratios in the treatment vs. the control group asexpected from the silencing effect of the NUC050 plasmid.

Because the average FF luciferase signals were higher in the NLJC050 vs.the NUC049 group, data were also analyzed by comparing the renilla:FFratios in subgroups of animals with similar levels of FF luciferaseexpression in both treatment groups. In the top or highest subgroup(most firefly luciferase) the ratio shift went from 33.5 for the controlNUC049 mice to 24.8 for the NUC050 mice (26%). Thus, there was a 26%reduction in the renilla:FF ratio in the subgroup of animals expressingthe highest amount of firefly luciferase. There were a total of 10 micein this group. The next highest subgroup showed an 8% difference andwent from a ratio of 30.8 to 28.4. There were only 5 mice in this groupor bucket. The remainder of the mice were in lower expressing groupswith lesser differences between the groups as expected but still somedifferences existed. The overall difference between the 2 groupscomparing all animals was 10%.

To assess the significance in the 10-26% reduction in renilla:FF ratioseen across groups and subgroups, we compared the reduction in ratio tothat in animals receiving both the target and effector plasmids togetherby direct hydrodynamic delivery. Direct hydrodynamic delivery of bothtarget and effector plasmids typically resulted in a maximal 30%silencing effect in this system. Therefore, as compared to a 30% maximaleffect, the 10-26% seen with this novel delivery mechanism appears quitesignificant and suggests that these experiments can serve as a basis fornovel methods and compositions for eiRNA and siRNA/shRNA mediated genesilencing via muscle delivery.

Example 5 In Vivo Electroporation Delivery of eiRNA: Dual LuciferaseModel

To test whether in vivo electroporation would be an efficient way todeliver eiRNA into skeletal muscle cells, the mice were firstanesthetized, the IM injection given and the electrodes placed into themuscle and the pulses are delivered. More specifically, C57B1/6 femalewere given an intramuscular (IM) injection in the tibialis muscle of oneleg of either NUC 050 (drug substance) or NUC 049 (negative control)formulation at a volume of 25 uL, concentration of 2.0 mg/ml. We used a3-pronged probe from Advisys, The Woodlands, Tex., and placed it on thetibialis muscle of the mouse leg. Two pulses of electricity wereadministered (see details in table below).

TABLE 1 Electroporator settings Pulse In Sequence: 1 2 Prewait (s): 4 1Pulse Width (ms): 52 52 Pulse Current (A): 0.1 0.1

Six days after dosing, all mice were given a hydrodynamic injection, or“challenge,” of 1 ug of the expression plasmid (NUC 060, dual-luciferaseHBV-fusion plasmid).

Five days after the hydrodynamic injection, all mice were sacrificed andtheir livers were dissected, frozen, and stored at −70 C. Livers werehomogenized with a mechanical homogenizer in a cell lysis buffer,centrifuged, and the supernatant was removed for analysis. Supernatantsamples from all livers were assayed for the presence of both Renillaand firefly luciferase proteins. The ratio of Renilla: fireflyluciferase (RLU) represents a normalized expression profile and servesas the output measure of the assay. The mean Renilla:firefly luciferaseratio of the NUC 050 group is compared to the mean ratio of the NUC 049group. This difference is represented as a net ratio value and a percentdifference:

$\overset{\%}{Difference} = {\frac{{{Mean}\mspace{14mu} {NUC}\mspace{14mu} 050} - {{Mean}\mspace{14mu} {NUC}\mspace{14mu} 049}}{{Mean}\mspace{14mu} {NUC}\mspace{14mu} 049} \times 100\%}$

The difference of the two means is tested for statistical significanceusing a nonparametric 2-sample Wilcoxon test for a p-value less than0.05.

The results indicate a 35% reduction in the fusion Renilla mRNA withrespect to the Firefly mRNA in the NUC050 group (See FIGS. 2 and 3 andTables 2 and 3). With respect to the results obtained with IM injection(non-electroporation) of NUC050, these results are consistent with thebetter muscle transfection when electroporation is used as the means fortransfecting muscle with the eiRNA vectors. This is also consistent witha liver delivery rate of siRNAs or shRNAs to hepatocytes approximatingand perhaps exceeding what is typically achieved by hydrodynamicinjection when the eiRNA is hydrodynamically injected first and then thedual luciferase vector challenge is given by hydrodynamic injection(HDI) later. We estimate based on this data that about 40%-60% ofhepatocytes have likely picked up one or more shRNAs encoded by NUC050.This is because each hydro injection typically transfects about 40-60%of hepatocytes. Transfection of hepatocytes following HDI is random andthe second HDI would hit about 40-60% of the cells that were transfectedby the first HDI.

TABLE 2 Individual Mouse Values (ratios) 300-1 300-2 # Nuc050 NucO49 116.8 19.3 2 17.7 22.4 3 18.6 25.4 4 18.7 26.0 5 20.0 26.1 6 20.6 30.8 721.4 32.9 8 21.8 34.9 9 23.2 36.6 10 23.3 39.7 11 23.5 42.1 12 23.7 44.013 27.4 44.9 Mean 21.3 32.7 Sd 2.97 8.49

TABLE 3 Additional Analysis Nuc050 Nuc049 Mean 21.28 32.69 StandardError 0.82 2.35 Median 21.43 32.90 Mode #N/A #N/A Standard Deviation2.97 8.49 Sample Variance 8.84 72.01 Kurtosis −0.09 −1.30 Skewness 0.340.00 Range 10.62 25.60 Minimum 16.78 19.30 Maximum 27.40 44.90 Sum276.70 425.01 Count 13 13

Example 6 In Vivo Electroporation Delivery of eiRNA Against HBV SurfaceAntigen

In this experiment, immunocompetent C57B16 mice were electroporatedintramuscularly with the anti-HBV eiRNA (NUC050) or with an irrelevanteiRNA (NUC049). Following electroporation, shRNAs encoded by the eiRNAvectors are transcribed to high copy levels in the electroporated muscle(2,000,000,000 copies of individual shRNA detected by QRT-PCR in pieceof electroporated muscle). Seven days post intramuscular electroporation(IM-EP), groups of mice were challenged by hydrodynamic injection withthree different doses of NUC054, an HBV expression vector encodingHBsAg. The doses were 10 ug, 5.0 ug and 2.0 ug NUC054. The hydrodynamicinjections also all contained identical amounts of a hAAT expressionvector as a marker for successful HDI, i.e., serum hAAT values are asurrogate marker of the amount of NUC054 transfection since bothplasmids were co-administered. The total amount of DNA was kept constantin each injection through the inclusion of an inert filler plasmid,pGL2-basic.

Two days, seven days and 16 days following challenge with NUC054, bloodwas collected from mice for measurement of HBsAg. If IM-EP of NUC050 isspecifically able to downregulate serum HBsAg (as compared to thecontrol NUC049), then transfected muscle must be able to relay plasmidspecified molecules such as shRNA/siRNA produced in the muscle tohepatocytes where RNAi of the HBV target mRNA occurs. This is becauseNUC054 which encodes the HBV mRNA is expressed in hepatocytesspecifically following HDI of NUC054 and if downregulation occurs, itmust occur in hepatocytes. HBsAg is made in hepatocytes and is secretedinto serum where it can be measured. As discussed further below, theresults indicate that NUC050 specifically and with statisticalsignificance downregulates the expression of HBsAg in mice that weregiven NUC050 via intramuscular electroporation.

Statistical Analysis

The serum surface antigen (sAg) concentrations 2 days post challengefrom mice that received NUC050 plasmid treatment and 2, 5 or 10 ug ofhydrodynamically injected sAg plasmid (NUC054) were compared to thegroup comprised of serum sAg concentrations on the same day from micethat received the control treatment NUC049 plasmid and either 2, 5, or10 ug of hydrodynamically injected NUC054 using a Wilcoxon two-sampleone-sided test application provided by Dr. Sam Litwin. The NUC050treatment resulted in statistically significantly lower serum sAg valuescompared to those from the control group (p=0.005).

The analysis above was performed for data obtained 7 days post challengeand the NUC050 treatment resulted in statistically significantly lowerserum sAg values when compared to the control group (p=0.002).

The serum hAAT values from mice that received NUC050 treatment and 2, 5or 10 ug of hydrodynamically injected sAg plasmid were matched with hAATvalues from mice that received the control NUC049 treatment and 2, 5 or10 ug of hydrodynamically injected NUC054 plasmid. Serum hAAT values area surrogate marker of the amount of NUC054 transfection since bothplasmids were co-administered. Data from unmatched animal pairs was notused. The day 2 sAg concentrations from mice that received NUC050plasmid treatment were compared to the matched group comprised of sAgconcentrations on the same day from mice that received the controltreatment NUC049 plasmid using a Wilcoxon paired-sample one-sided testapplication provided by Dr. Sam Litwin. The results of the test showedthat the NUC050 treatment resulted in statistically significantly lowersAg concentrations compared to the control group (p=0.011).

The serum hAAT values from mice that received NUC050 treatment and 10 ugof hydrodynamically injected sAg plasmid were matched with hAAT valuesfrom mice that received the control NUC049 treatment and 10 ug ofhydrodynamically injected NUC054 plasmid. Serum hAAT values are asurrogate marker of the amount of sAg transfection since both plasmidswere co-administered. Data from unmatched animal pairs was not used. ThesAg concentrations on day 2 from mice that received NUC050 plasmidtreatment were compared to the matched group comprised of sAgconcentrations on the same day from mice that received the controltreatment NUC049 plasmid using a Wilcoxon paired-sample one-sided testapplication provided by Dr. Sam Litwin. The results of the test showedthat the NUC050 treatment resulted in sAg concentrations that werestatistically significantly (p=0.009) less than those from the controlNUC049 plasmid group. See Table 4.

TABLE 4 Sample Test p-value Combined day 2 data Wilcoxon 2 sample 0.005Combined day 7 data Wilcoxon 2 sample 0.002 Combined day 2 data matchedWilcoxon paired 0.011 10-ug day 2 data matched Wilcoxon paired 0.009

Example 7 Quantitation of siRNA Expressed in Muscle

The purpose of this experiment was to measure the amount of siRNAexpressed in muscle from an eiRNA plasmid following intramuscularelectroporation. For our hypothesis of transfected muscle cell to act asa depot to deliver siRNA/shRNA or DNA to distal sites, the muscle mustfirst be transfected. If muscle is secreting or exporting vesiclescontaining siRNA/shRNA, then the more of these molecules that areexpressed, the more siRNA/shRNA will be shipped out of the cell, and ahigher delivery to distal sites is expected.

In this experiment, mice were electroporated intramuscularly withNUC050. NUC050 expresses four HBV-specific shRNAs that are processedinto siRNAs. For this experiment, only one of the siRNAs encoded wasmeasured. Following electroporation, the area of muscle electroporatedwas harvested and snap frozen. RNA was subsequently extracted and one ofthe siRNAs expressed from NUC050, sil737, was measured by QRT-PCR.

QRT-PCR for detection of expressed siRNA's targeting HBV includingsi1737, si1907 and si2791 was performed utilizing the protocol describedby Caifu Chen, et al. in (Chen Caifu/ABI Method: Nucleic Acid Research,2005, 33(20):el 79). Sil737 served as an example of the protocol. Theprocedure involves a reverse-transcription reaction to generate cDNAfrom the siRNA. This is done primarily by the use of a loop primer,which matches 8 nucleotides of the siRNA sequence of 1737. (FIG. 4) Thisprotocol utilizes a stem-loop primer for generating the cDNA for PCRfrom expressed siRNA. This method was shown to provide better RTspecificity and efficiency than linear primers due to the base stackingof the stem of the primer, which enhances the thermal stability of theresulting RNA-DNA heteroduplex. The spatial constraint of the loopprimer also improves assay specificity compared to linear primers asshown by Chen.

RNA for reactions was collected by the Invitrogen Trizol protocol (cat#15596-026). RNA from muscle tissue was extracted in Trizol usinghomogenization. The RT reaction was done in a 15 ul reaction volume withfinal concentrations of 10 mM MgCl₂, Ix GeneAmp PCR buffer (AppliedBiosystems cat. # N808-0010), 50 nM loop primer, 0.26 mM dNTP, 3.33 U/μlMultiScribe Reverse Transcriptase (Applied Biosystems cat #4319983),0.26 U/μl GeneAmp RNase Inhibitor (Applied Biosystems cat. # N808-0119).Reactions were incubated in an MJ Research PTC-200 Peltier ThermalCycler for 30 min at 16° C., 30 min at 42° C. and 5 min at 85° C. ThecDNA was then added to the Q-PCR reaction with a higher concentration ofthe forward primer. The excess forward primer preferentially binds andgenerates more antisense cDNA. The FAM probe for Q-PCR then binds theantisense cDNA along with the reverse primer, thus starting the Q-PCRdetection. The Q-PCR reaction was done in 20 μl reaction volume with 2μl of RT product input, and final concentrations of Ix Taqman UniversalMaster Mix No Amperase UNG (Applied Biosystems cat #4324018), 1500 nMforward primer, 750 nM reverse primer, 200 nM FAM probe. Reactions wererun in an Applied Biosystems Real-Time PCR System 7300 for 40 cycles of95° C. for 15 sec and 60° C. for 1 min using a FAM detector. Primer andprobe sequences were as follows:

The sequences of si 1737 (underlined strands) and its loop primer arelisted below:

(SEQ ID NO: 1) 5′-GGAUUCAGCGCCGACGGGACG-3′ (SEQ ID NO: 2)3′-TGCCCTGCGAGTTG-ACTTAACGG CTGAGGTGCTGTGGTCAACTC-5′

After RT reaction, a cDNA as following will be synthesized:3′-CCTAAGTCGCGGCTGCCCTGCGAGTTG-ACTTAACGGCTGAGGTGCTGTGGTCAACTC-S′ (SEQ IDNO: 3)

In Q-PCR reaction, higher concentration of forward primer (pink strand)preferentially makes more antisense cDNA (the blue strand):

The antisense cDNA will be used for probe and reverse primer binding:

(SEQ ID NO: 7) 5′CAGCTGGGA-GGATTCAGCGCCGAC-S′ (SEQ ID NO: 8)5′CAGCTGGGA-GGATTCAGCGCCGACGGGACGCTCAAC-TGAATTGCCGACTCCACGACACCAGTTGAG-S′ (SEQ ID NO: 9)Q-TGCCCTGCGAGTTG-ACTT-Fam (SEQ ID NO: 10) 3′-CTGAGGTGCTGTGGTCAACT-S′

Si 1737 chemically synthesized by IDT, Ames Iowa was used to generate astandard curve and for use in spike recovery.

All four electroporated muscles from study ND311 that were injected withNUC050 and APL050 showed expression levels of sil737 between 8.0 e+006to 22 e+009 siRNAs. Electroporated muscles injected with the sAgexpression vector alone, APL050, did not yield any detection of siRNA aswe predicted. Naïve muscle was also negative for siRNA expression aspredicted. For results, see Table 5 below.

TABLE 5 sample name Detector Task Ct quantity 6.00E+08 1737 Standard12.93 6.00E+08 6.00E+07 1737 Standard 15.55 6.00E+07 6.00E+06 1737Standard 19.52 6.00E+06 6.00E+05 1737 Standard 22.51 6.00E+05 6.00E+041737 Standard 26.05 6.00E+04 ND311 APL050 1737 Unknown undet. 1 A muscleND311 APL050 1737 Unknown undet. 1 B muscle ND311 APL050 1737 Unknownundet. 1C muscle ND311 APL050 1737 Unknown undet. 1 D muscle ND311ALPO5O + 1737 Unknown 19.04 7.24E+06 NucO5O 2A muscle ND311 ALP050 +1737 Unknown 19.88 4.04E+06 Nuc050 2B muscle ND311 ALPO5O + 1737 Unknown14.95 1.24E+08 NucO5O 2C muscle ND311 ALP050 + 1737 Unknown 15.976.10E+07 Nuc050 2D muscle Na

ve muscle 1737 Unknown undet. Negative control H20 1737 Unknown undet.Negative control H20 1737 Unknown undet.Note 1. RNA extracted from muscle was resuspended in a total volume of12 ul. 5 ul of this extract was added to the initial RT reaction ofwhich the total volume was 15 ul. 2 u/l/15 ul of the RT-reaction wasused for a PCR reaction. This means the PCR values for siRNA copy numberwere multipled by 18 to get the copy number of si 1737 in the muscleextracts. For example, the results obtained and displayed as data in theabove table were multiplied by 18 to get the total copy number of sil737in the extracted muscle sample.

Example 8 Electron Micrographs of Muscle Blebs

Photos of published muscle blebs stained immunologically for B-galectin(FIG. 5, panels B, C and D) were compared with a time lapsed photo bythe present inventors of a myoblast secreting blebs (panel A). Prior totaking the picture, L6 cells (which contain myoblasts in culture thatdifferentiate into myotubes) were transfected with an EGFP expressionplasmid to see if we could see blebs and if so, whether they containEGFP. If so, this indicates that blebs pack up cytosolic content intotheir interior. The picture in FIG. 5, panel A shows transfectedmyoblasts with bleb like structures that are fluorescent.

Example 9 Prophylactic Inhibition of HBV sAg Gene Expression In Vivo byIntramuscular Injection

This example shows that subcutaneous injection of an eiRNA plasmidexpressing siRNAs specific for Hepatitis B causing the distaldownregulation of mRNAs containing Hepatitis B target sequences in liverhepatocytes. The results of the data would indicate that subcutaneousinjection of eiRNA molecules/siRNA molecules and likely intradermalinjection as well can mediate the downregulation of gene(s) inhepatocytes, not limited to the HBV sAg gene exemplified here. Themolecules transferred to distal sites could be siRNA/shRNA, the eiRNAplasmid DNA or the RNA charged RNA Induced Silencing Complex. AnysiRNA/shRNA and/or DNA would therefore be predicted to transfer.Although this model is prophylactic, since these results show that theactive agent is transferring to hepatocytes, a therapeutic subcutaneousinjection is also predicted to work.

Experiment Background

In the prophylactic model, the drug molecule (plasmid expressing theshRNA silencing molecule) is administered before the target molecule(plasmid expressing an RNA to be silenced) is administered.

In all experiments “NUC050” refers to an eiRNA plasmid expression vectorthat expresses four different short hairpin dsRNAs (shRNAs) specific toHBV (see e.g., WO 2006/033756, plasmid pHB4, FIG. 9), which mediatessilencing of an HBV-Luciferase fusion vector, “NUC060”, e.g., asdescribed in WO 04076629/US 2006/0263764. “NUC049” is a negative controleiRNA plasmid (which expresses a mutated version of a single shRNAderived from NUC050).

The target plasmid contains two separate genes encoding luciferases fromtwo different organisms, Firefly (FF) and jellyfish (Renilla or “JF”).See WO 04076629: Methods and constructs for evaluation of RNAi targetsand effector molecules. Because each produces a different wavelength ofluminescence upon hydrolysis of luciferin substrate, the two signals canbe measured in the same sample. The Renilla luciferase mRNA isengineered as a fusion mRNA with sequence elements present in the HBVgenome, such that the fusion mRNA becomes a target for the NUC050 eiRNAplasmid, and the signal from Renilla is a measure of the gene silencingeffect of NUC050. Activity of NUC050 will therefore result in areduction of the Renilla luciferase signal even though it targets HBVsequences because the HBV sequences are present at the end of theRenilla mRNA. The FF luciferase signal serves as a normalizationstandard to correct for overall variability in target plasmiddelivery/transfection, because it is not subject to down modulation bythe eiRNA effector, but is dependent on the same plasmid for expression.Thus, taking the ratio, JF:FF luciferase normalizes for theliver-directed expression of plasmid. In cases where an insufficient FFluciferase is observed, it can be concluded that the plasmid was notdelivered, and therefore the sample is not valid for analysis.

In vivo subcutaneous delivery of eiRNA: dual luciferase model

C57B1/6 female mice were given a subcutaneous (SC) injection of eitherNUC 050 (drug eiRNA substance) or NUC 049 (negative control eiRNAsubstance) formulation at a volume of 200 μl, concentration of 1.5mg/ml. The formulations consisted of DNA in an isotonic saline solution.Six days after dosing, all mice were given a hydrodynamic injection(HDI), or “challenge,” of 1 ug of the expression plasmid (NUC 060,dual-luciferase HBV-fusion plasmid). HDI is a mechanism that allowsselective transfection of liver hepatocytes. [00143] Five days after thehydrodynamic injection (day 11), all mice were sacrificed and theirlivers were dissected, frozen, and stored at −70 C. Livers werehomogenized with a mechanical homogenizer in a cell lysis buffer,centrifuged, and the supernatant was removed for analysis. Supernatantsamples from all livers were assayed for the presence of both Renillaand Firefly luciferase proteins. The ratio of Renilla:Firefly luciferase(RLU) represents a normalized expression profile and serves as theoutput measure of the assay. The mean Renilla:Firefly luciferase ratioof the NUC 050 group is compared to the mean ratio of the NUC 049 group.This difference is represented as a net ratio value and a percent

$\overset{\%}{Difference} = \frac{{{Mean}\mspace{14mu} {NUC}\mspace{14mu} 050} - {{Mean}\mspace{14mu} {NUC}\mspace{14mu} 049}}{{Mean}\mspace{14mu} {NUC}\mspace{14mu} 049}$

difference:

The difference of the two means is tested for statistical significanceusing a nonparametric 2-sample Wilcoxon test for a p-value less than0.05.

In this experiment, 15 animals were used for each treatment group.Levels of FF luciferase were acceptable for 8/15 animals in both theeiRNA (NUC050) group and the control (NUC049) group. A box-and-whiskerplot of Renilla:FF activity ratios in liver cells shows a clear andstatistically significant decrease of the Renilla Luciferase-HBV mRNAfusion target in the treatment vs. the control group. Mean levels of thetarget mRNA are reduced by 16.7% in the treatment group (see FIGS. 8 and9, Tables 6 & 7).

The observed silencing of the target mRNA indicates that either theplasmid DNA, the expressed eiRNA and/or its processed product (ie. theds siRNA or RNA charged RNA Induced Silencing Complex) have beentransported from the subcutaneous injection site to hepatocytes in theliver. This transport may be mediated by cells at the injection site.Regardless of the mechanism involved however, these results show thatsubcutaneous injection of an eiRNA expressing plasmid can reduce thelevels of target mRNA at a distal site, in this case liver hepatocytes.

TABLE 6 Individual Mouse Values (ratios) 360-1 360-2 (Nuc050) (Nuc049) 112.0 12.3 2 12.2 14.4 3 12.3 17.6 4 13.3 17.7 5 16.1 18.2 6 16.69 18.3 717.2 21.8 8 18.5 22.1 Mean 14.8 17.8 Sd 2.6 3.3

TABLE 7 Additional Analysis 360-1 360-2 (Nuc050) (Nuc049) Mean 14.817.79 Standard Error 0.93 1.17 Median 14.68 17.94 Mode #N/A #N/AStandard Deviation 2.62 3.30 Sample Variance 6.88 10.88 Kurtosis −2.10−0.18 Skewness 0.17 −0.34 Range 6.47 9.78 Minimum 12.03 12.28 Maximum18.50 22.06 Sum 118.47 142.30 Count 8 8

Example 10 Therapeutic Inhibition c Subcutaneous Adoptive Transfer ofNUC050-Transfected Cells

The NUC050 vector encodes four different shRNA molecules which targetvarious portions of the hepatitis B genome for degradation via thecellular RNAi mechanism. Three of these target the HBsAg regionscontained in the target vector preadministered to the mice.

In the therapeutic model, animals receive exogenous RNA target in theform of a plasmid expressing the surface antigen coding sequence of HBVvia hydrodynamic tail vein injection. Hydrodynamic injection is a methodusing a large volume with rapid injection time, to preferentially directthe DNA plasmid to the liver where it is expressed.

To obtain mice expressing a target gene in liver, HBsAg (surfaceantigen) cDNA was placed under the control of a liver-specific promoterin a commercially available plasmid vector (pLIVE). On Day −7, thisvector was injected hydrodynamically into an immunodeficient strain ofmice {NOD. CBI 7-Pkrdc^(scld)/J). In this way, DNA is largely localizedto liver hepatocytes and the tissue-specific promoter further restrictsexpression of the sAg mRNA to these cells. Since the mice areimmunodeficient, they are able to express the target gene for longperiods of time (greater than a month) because immune response to theforeign protein encoded by the target gene will not be made.

Four days following target plasmid administration (Day −3), the micewere bled to determine levels of circulating HBsAg in serum. On Day 0,mice were subcutaneously injected with a suspension of RD(rhabdomyosarcoma) cells (RD; ATCC CCL-135) which had been transfectedwith NUC050 within a total volume of 0.2-0.4 ml. A control group of micewas treated similarly with a suspension of RD cells which had beentransfected with the negative control NUC049 plasmid.

The NUC050 and NUC049 cell suspensions were prepared identically asfollows:

Briefly, RD cells were plated in TI 50 flasks (150 cm²) at a density of5×10⁶ cells per flask and incubated @ 37° C., 5% CO₂. One day later theywere transfected with either NUC050 vector or NUC049 negative controlvector (28 ug/flask, respectively) mixed with the Roche Fugene™ 6transfection reagent (cat. #11814443001). Cells were roughly 65%confluent at the time of transfection. After transfection mix was added,the RD cells were incubated overnight @ 37° C., 5% CO₂. The next day thecells were rinsed in I× Dulbecco's Phosphate Buffered Saline (DPBS) andthe medium was changed to fresh Dulbecco's Modified Eagle's Medium(DMEM) with 10% FBS. Three days post transfection, RD cells were rinsedin sterile DPBS and trypsinized to liberate them from the flask surface.Cells were counted and multiple aliquots of 1×10 cells were rinsed twicein sterile DPBS and pelleted by centrifugation. The RD cells were thenresuspended in 4.5 ml sterile DPBS. A 0.2-0.4 ml cell suspensioncontaining approximately 8×10⁶ cells was then injected subcutaneouslyinto each NOD.CB17-ZWc*^(nd)/J (NOD/SCID) mouse in the right flank,using a 23 gauge needle.

Animals were subsequently bled again on Days 6 and 11 post eiRNA vectoradministration, respectively and levels of serum HBsAg were determinedusing a commercially available HBsAg ELISA (Bio-Rad, Hercules, Calif.HBsAg 3.0 EIA cat#. 32591). Animals are scheduled to be bled again onDays 18 and 25 days post eiRNA vector administration as well withsubsequent HBsAg level measurement. From 9 to 11 mice were used for eachtreatment group and the results are presented as HBsAg percent ofinitial HBsAg values (pre-treatment). The ability of the adoptivelytransferred NUC050 vector treated cells to decrease HBsAg expression wasestimated by calculating, for each bleed day, a normalized differencevalue of average HBsAg levels for control minus experimental groups.Normalization was done by calculating the percent of HBsAg for eachsubsequent bleed day, relative to the HBsAg values on pretreatment.

Day 6 and Day 11 show difference averages of −8% and −18% respectively,between NUC050 and NUC049 (FIG. 10 and Table 8). (These values reflectunderlying normalized average values of 124% and 132% prebleed(pretreatment) values for NUC050 and NUC049 respectively for Day 6. Thenormalized average values compared to Prebleed values were 105% (NUC050)vs. 123% (NUC049) for Day 11). Based on this trend and historical datatrends within this animal model, we anticipate increasing differencesbetween treatment and control groups in the subsequent time points (Day18 and Day 25).

Exogenously transfected cells e.g. muscle cells as described hereincould also be cultured and their culture medium used as a source ofRNA-containing “blebs” or exovesicles for use as described elsewhereherein. One of skill in the art will also recognize that the methodsdescribed can be utilized for heterologous or autologous transplant intoa recipient mammal of exogenously transfected cells such as muscle cellsfor use as a source of biologically active RNA effector moleculesdelivered to distal cells including liver cells such as hepatocytes.

TABLE 8 Normalized HBsAg Levels (% prebleed) time pt Nuc049 Nuc050Difference Day 6 132.2 123.8 −8.4 Day 11 123.0 105.4 −17.6

Example 11 In Vivo Electroporation Delivery of eiRNA Against EndogenousInterleukin-12 (IL-12)

In this experiment, immunocompetent BALB/cJ mice are administered ananti-IL-12 eiRNA vector or an irrelevant, negative control eiRNA vectorvia intramuscular (IM) injection concomitantly with electroporation(EP), (IM-EP). The anti-IL-12 eiRNA plasmid vector encodes a shRNAmolecule driven by a 7SK promoter, which targets the p40 subunit of themouse IL-12 mRNA sequence for degradation via the sequence specificcellular RNAi mechanism. Following IM-EP administration, shRNA moleculesencoded by the eiRNA vectors are transcribed to high copy levels in theelectroporated muscle (2,000,000,000 copies of individual shRNAdetectable by QRT-PCR in piece of electroporated muscle).

One day, four days, seven days, and 11 days following dosing with theanti-IL-12 and control eiRNA vectors, blood is collected from mice formeasurement of IL-12 using a commercially-available ELISA (R&D Systems).If IM-EP delivery of the anti-IL-12 eiRNA vector is specifically able todownregulate endogenous serum IL-12 (as compared to the control vector),then transfected muscle must be able to relay plasmid expressedmolecules such as shRNA/siRNA produced in the muscle to at least some ofthe various distal immune cells (macrophages, monocytes, dendriticcells, and B cells) which express IL-12 and where RNAi of the IL-12target mRNA occurs. Because IL-12 is expressed by multiple cell typesthroughout the body, significant downregulation would be likely tosignal substantial delivery to multiple sites where such cells reside.In our described study, we expect intramuscular injection of theanti-IL-12 eiRNA vector with electroporation (IM-EP) in mice to resultin sustained downregulation of endogenous serum IL-12 over all timepoints. We expect that alternative methods of delivery to muscle cells{e.g., vascular delivery with increased pressure to mammalian limbmuscle as described elsewhere herein) would also achieve downregulationof a target nucleic acid such as IL-12 in distal non-muscle cellsincluding immune cells.

All publications, patents and patent applications discussed herein areincorporated herein by reference. While in the foregoing specificationthis invention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

What is claimed:
 1. A method of delivering at least one RNA to a targetcell in an animal comprising: transfecting a first cell in vitro with anucleic acid encoding said RNA, isolating at least one exovesicle fromsaid first cell wherein said exovesicle contains said RNA, andcontacting said target cell with said exovesicle.
 2. The method of claim1, wherein said RNA is a dsRNA.
 3. The method of claim 1, wherein saidfirst cell is a muscle cell or a skin cell.
 4. The method of claim 1,wherein a nucleic acid expressing a transmembrane or surface ligandspecific for said target cell is co-transfected with said nucleic acidencoding said RNA.
 5. The method of claim 4, wherein said nucleic acidexpressing said transmembrane or surface ligand and said nucleic acidencoding said dsRNA are encoded on a single plasmid.
 6. A method ofdelivering at least one RNA to a target organ or tissue in an animalcomprising transfecting at least one first cell ex vivo with a nucleicacid encoding said RNA, and introducing said transfected cell into saidanimal, wherein said introduction results in said RNA being delivered toat least one second cell in said target organ or tissue, wherein saidtarget organ or tissue is distal from the site of introduction of saidtransfected first cell.
 7. The method of claim 6, wherein said RNA is adsRNA.
 8. The method of claim 6, wherein said first cell is a musclecell or a skin cell.
 9. The method of claim 6, wherein a nucleic acidexpressing a transmembrane or surface ligand specific for said targetcell is co-transfected with said nucleic acid encoding said RNA.
 10. Themethod of claim 9, wherein said nucleic acid expressing saidtransmembrane or surface ligand and said nucleic acid encoding saiddsRNA are encoded on a single plasmid.